Pharmaceuticals and methods for treating hypoxia and screening methods therefor

ABSTRACT

Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. Nos.10/101,812, filed Mar. 19, 2002; which claims priority to U.S.Application No. 60/277,425, filed Mar. 20, 2001; 60/277,431, filed Mar.20, 2001; 60/277,440, filed Mar. 20, 2001; 60/332,493, filed Nov. 9,2001; 60/332, 334, filed Nov. 9, 2001; 60/345, 200, filed Nov. 9, 2001;60/345,131 filed Dec. 20, 2001, 60/342,598, filed Dec. 20, 2001; and60/345,132, filed Dec. 20, 2001 each of which are incorporated herein byreference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with U.S. government support under ______grants ______. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The invention relates to drug discovery. The invention featuresthe use of light emitting proteins as tools for diagnosis, drugscreening and discovery, and as pharmaceuticals for in vivo treatment.

[0004] A key advance in the biomedical arts has been the discovery ofbioluminescent protein moieties, e.g., green fluorescent protein (GFP)and luciferase, which can be expressed in diverse mammalian cell typesand thus act as detectable signals for biological signal transductionpathways and events. An increased understanding of how diversebiological processes are regulated by the actions of cellular enzymes,e.g., kinases, proteases, and ubiquitin ligases, has also been emerging.Alterations in the activity of these enzymes may underlie the initiationand/or progression of diseases such as cancer.

[0005] Methods of detecting biological activities and substances usingbioluminescent proteins have recently been developed. For example,protein phosphorylation events can be detected using fusion proteinscontaining GFP (see, e.g., U.S. Pat. No. 5,958,713) or luciferase,aequorin and obelin (see, e.g., U.S. Pat. No. 5,683,888).Light-generating moieties have been introduced into mammals tospecifically localize events such as parasite infection (see, e.g., U.S.Pat. No. 5,650,135).

[0006] How cells sense changes in ambient oxygen is a central problem inbiology. In mammalian cells, lack of oxygen, or hypoxia, leads to thestabilization of a sequence-specific DNA-binding transcription factorcalled HIF (hypoxia-inducible factor), which transcriptionally activatesa variety of genes linked to processes such as angiogenesis and glucosemetabolism.

[0007] Tissue ischemia is a major cause of morbidity and mortality.Ischemia can result from chronic hypoxia brought on by lack of bloodsupply to the tissue occurring from, for example, stroke, deep veinthrombosis, pulmonary embolus, and renal failure. Ischemic tissue isalso found in tumors.

[0008] HIF binds to DNA as a heterodimer consisting of an alpha subunitand a beta subunit (also called aryl hydrocarbon receptor nucleartranslocator or ARNT). The alpha subunit is rapidly polyubiquitinatedand degraded in the presence of oxygen whereas the beta subunit isconstitutively stable. von Hippel-Lindau (VHL) disease is a hereditarycancer syndrome characterized by the development of highly vasculartumors that overproduce hypoxia-inducible mRNAs such as vascularendothelial growth factor (VEGF). The product of the VHL tumorsuppressor gene, pVHL, is a component of multiprotein complex thatcontains elongin B, elongin C, Cul2, and Rbx1. This complex bearsstructural and functional similarity to SCF (Skp1/Cdc53 or Cullin/F-box)ubiquitin ligases. In the presence of oxygen pVHL binds directly to HIFαsubunits and targets them for polyubiquitination and destruction. Cellslacking functional pVHL can not degrade HIF and thus overproduce mRNAsencoded by HIF-target genes.

[0009] Progress has also been made in recent years towards understandingthe molecular mechanisms in control of cell proliferation. Aberrant cellproliferation is a hallmark event in the onset and progression ofdiseases such as cancer. Progression through the mammalian cell-cycle islinked to the orchestrated appearance and destruction of cyclins.Different cyclins are associated with different cell-cycle transitions.For example, cyclin E is active in late G1 and early S-phase, cyclin Ais active in S-phase, and cyclin B is active in mitosis. Cyclins bind tocyclin-dependent kinases (cdks). In this context, cyclins activate thecatalytic activity of their partner cdk(s) and also play roles insubstrate recognition.

[0010] Some transcriptional regulatory proteins, such as the pRBhomologs p107 and p130, the E2F family members E2F1, E2F2, and E2F3, thetranscriptional coactivator p300, and NPAT (nuclear protein mapped tothe AT locus) form stable complexes with cyclin A/cdk2 and/or cyclinE/cdk2. All of these proteins bind directly or indirectly to DNA. Thus,such complexes might serve as vehicles for increasing the concentrationof cyclin A/cdk2 or cyclin E/cdk2 at certain sites within the genome. Assuch, cyclin A/cdk2 and cyclin E/cdk2 might play relatively direct rolesin processes such as transcription and DNA replication. These twoprocesses are fundamental in normal cell proliferation and are perturbedduring aberrant cell proliferation, such as in cancer.

SUMMARY OF THE INVENTION

[0011] The invention relates in part to the discovery oflight-generating fusion proteins (or a cell expressing thelight-generating fusion protein), wherein the light-generating fusionprotein features a ligand binding site and a light-generatingpolypeptide moiety. The light-generating fusion protein (“LGP”) has afeature where the light generation of the light-generating polypeptidemoiety changes upon binding of a ligand at the ligand binding site. Theligand may be active in an environment only under certain conditions,e.g., in a hypoxic state, such that the light-generating fusion proteinis “turned” on or off only under such conditions.

[0012] The light-generating fusion proteins of the invention may be usedfor screening for modulators of activity or latency of (orpredisposition to) disorders such as hypoxia, cancer, diabetes, heartdisease or stroke. For example, a test compound may be administered to atest animal at increased risk for such a disorder, wherein the testanimal recombinantly expresses a light-generating fusion protein,allowing for localization of the light-generating fusion protein,detecting the luminescence of the light-generating polypeptide moiety inthe test animal after administering the test compound, and comparing theluminescence of the light-generating polypeptide moiety in the testanimal with the luminescence of the light-generating polypeptide moietyin a control animal not administered the test compound. A change in theactivity of the light-generating polypeptide moiety in the test animalrelative to the control animal indicates the test compound may be amodulator of, e.g., prolyl hydroxylase.

[0013] The effects of an anti-hypoxic compound in vivo may be determinedin another embodiment, by administering to a subject, e.g., mammalian, alight-generating fusion protein comprising an ubiquitin ligase bindingsite and a light-generating polypeptide moiety or a cell expressing thelight-generating fusion protein, allowing for localization of thelight-generating fusion protein or cell in hypoxic tissue in thesubject; and measuring the luminescence of the localizedlight-generating fusion protein from the hypoxic tissue.

[0014] Methods in accordance with the invention are also provided fordetermining the effects of an anti-cell proliferation compound understudy in a mammalian subject, by administering to a test subject alight-generating fusion protein containing a cyclin/cdk binding site ora cell expressing the light-generating fusion protein of the invention,allowing for localization of the fusion protein or cell, measuringluminescence from the localized light-generating fusion protein; andimaging the localized light-generating fusion protein, therebydetermining the effects of the anti-cell proliferation compound.

[0015] Cyclin/cdk activity may be assayed in another embodiment of theinvention wherein a test sample is contacted with a light-generatingfusion protein comprising a cyclin/cdk binding site and alight-generating polypeptide moiety; and thereafter the presence oramount of cyclin/cdk activity in said test sample is determined bymeasuring the luminescence of the test sample.

[0016] The invention yet further relates to DNA constructs, including anisolated DNA encoding a modified LGP wherein one or more amino acidshave been substituted, inserted or deleted to provide a ligand bindingsite such as a ubiquitin ligase or protease recognition site, whereinfluorescence of the LGP changes upon binding of a ligand to the ligandbinding site.

[0017] The invention also relates to light-generating fusion proteinshaving a ligand binding site, such as a ubiquitin ligase binding site,or a HIF1α polypeptide moiety; and a light-generating polypeptidemoiety. Another embodiment features a light-generating fusion proteinwith a cyclin/cdk binding site, a suicide protein polypeptide moiety,and a light-generating polypeptide moiety; and a light-generating fusionprotein comprising a protein dimerization domain and a light-generatingprotein moiety. In a preferred embodiment, the light-generating fusionproteins have the property that upon ligand binding to the ligandbinding site, the luminescence of the light-generating polypeptidemoiety is changed without altering the phosphorylational state of thefusion protein.

[0018] The invention further relates to fusion proteins including aHIF1α polypeptide moiety or cyclin/cdk binding site, and a suicideprotein polypeptide moiety. A light-generating polypeptide moiety mayoptionally be included. These protein constructs may be used toselectively target certain cells, e.g., hypoxic tumor cells, fordestruction. For example, the invention includes methods of treatinghypoxic or ischemic disorders by administering to a subject an effectiveamount of a fusion protein comprising a HIFα polypeptide moiety having abinding affinity for prolyl hydroxylase, and a suicide polypeptidemoiety, such that the hypoxic or ischemic disorder is treated. Methodsof killing hypoxic tumor cells, wherein an effective amount of thefusion/suicide protein is administered to a subject, such that thehypoxic tumor cells are killed; and methods of treatingcell-proliferating disorders by administering to a subject an effectiveamount of a fusion protein comprising a cyclin/cdk binding site and asuicide protein polypeptide moiety, such that the cell-proliferatingdisorder is treated, are also contemplated.

[0019] The treatment of cell-proliferating disorders may be monitored byan embodiment of the invention, e.g., by administering to a subject aneffective amount of a fusion protein comprising a HIFα polypeptidemoiety having a binding affinity for prolyl hydroxylase, a suicidepolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of the light-generating fusion protein changes uponbinding of prolyl hydroxylase to the HIFα polypeptide moiety, such thatthe cell-proliferating disorder is treated, and monitoring the abilityof the fusion protein to inhibit cell proliferation by measuring thelight generated by the light-generating fusion protein. Alternately,treatment of cell-proliferating disorders may be monitored byadministering to a subject an effective amount of a light-generatingfusion protein comprising a cyclin/cdk binding site, a suicide proteinpolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of the light-generating fusion protein changes uponbinding of a cyclin to the cyclin/cdk binding site, such that saidcell-proliferating disorder is treated; and monitoring the ability ofthe fusion protein to inhibit cell proliferation by measuring the lightgenerated by the light-generating fusion protein.

[0020] Other embodiments of the invention include a cyclin/cdk bindingsite and a light-generating polypeptide moiety; and a protease bindingsite and a light-generating protein moiety. Antibodies specific for aprotein complex comprising HIF1α and pVHL are also detailed as withinthe present invention.

BRIEF DESCRIPTION OF THE DRAWING

[0021]FIG. 1 is a schematic representation of different fusion proteinsof the present invention.

[0022]FIG. 2 is a second schematic representation of different fusionproteins of the invention.

[0023]FIG. 3 shows pVHL binding to a modified form of HIF.

[0024]FIG. 4 shows pVHL binding to a HIF1α-derived peptide if Leu562 andPro564 are intact.

[0025]FIG. 5 shows ubiquitination and degradation of HIF linked toLeu562 and Pro 564.

[0026]FIG. 6 depicts proline hydroxylation linked to pVHL-binding.

[0027]FIG. 7 illustrates pVHL specifically recognizing HIF1α withhydroxylated proline 564.

[0028]FIG. 8 illustrates the production of TETr-cyclins A and E.

[0029]FIG. 9 shows DNA bound cyclins A and E differentially affectingtranscription.

[0030]FIG. 10 illustrated transcriptional regulation by cyclins A and Edependent upon DNA binding.

[0031]FIG. 11 depicts cyclin box is required for transcriptionalrepression by DNA bound cyclin A.

[0032]FIG. 12 illustrates that transcriptional activation by cyclin E islinked to its ability to bind to cdk2 and interact with substrates.

[0033]FIG. 13 shows that transcriptional activation by DNA bound cyclinE depends on cdk2 catalytic activity.

[0034]FIG. 14 shows transcriptional effects mediated by cell-cycledependent changes in endogenous cyclins E and A.

[0035]FIGS. 15-18 illustrate the results obtained in Example 2.

[0036]FIG. 19 illustrates the wild type sequence of HIF1α, Accession No.Q16665 (SEQ ID NO: 1X).

DETAILED DESCRIPTION OF THE INVENTION

[0037] Definitions

[0038] “Light-generating” or “luminescent” includes the property ofgenerating light through a chemical reaction or through the absorptionof radiation, including phosphorescence, fluorescence, andbioluminescence.

[0039] “Light” includes electromagnetic radiation having a wavelength ofbetween about 300 nm and about 1100 nm.

[0040] “Non-invasive” methods for detecting localization in a subjectdoes not include largely invasive methods such as conventional surgeryor biopsy.

[0041] “Light-generating fusion protein” includes proteins of theinvention having a light-generating or luminescent portion, i.e., alight-generating polypeptide moiety and a ligand binding site. Ingeneral, when a ligand of interest binds to the ligand binding site ofthe light-generating fusion protein, the light-generating properties ofthe light-generating polypeptide moiety change, either going from “dark”to “light”, or vice versa.

[0042] “Light-generating polypeptide moiety” includes any protein knownto those of ordinary skill in the art to provide a readily detectablesource of light when present in stable form. Non-limiting examplesinclude light-generating proteins described in U.S. Pat. Nos. 5,683,888,5,958,713, and 5,650,135, e.g., ferredoxin IV, green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, bluefluorescent protein, the luciferase family and the aequorin family. In apreferred embodiment, the light-generating polypeptide moiety is aprotein such as green fluorescent protein, red fluorescent protein,yellow fluorescent protein and blue fluorescent protein.

[0043] “Colinear effector site” includes regions of the light-generatingpolypeptide moiety that, when acted on by events subsequent to ligandbinding, cause the light-generating polypeptide moiety to change itspresent light-emitting state (i.e., on or off). These regions making upthe colinear effector site may do this by, e.g., conformationaldistortion, chemical modification, e.g., ubiquitination of a residue orresidues in the colinear effector site, or by cleavage of a portion ofall or part of the colinear effector site.

[0044] “Having binding character for prolyl hydroxylase” refers to aproperty making HIFα polypeptide moieties suitable for, e.g., screeningmethods of the invention and includes HIF polypeptide sequences suitableor adapted for prolyl hydroxylase binding as well as native or wild-typeHIF sequences to which pVHL has recognized and bound.

[0045] “Having binding character for ubiquitin ligase” refers to aproperty making HIFα polypeptide moieties suitable for, e.g., screeningmethods of the invention, e.g., including native HIF polypeptidesequences having hydroxylated proline residues hydroxylated by prolylhydroxylase, or, e.g., “HIF1α polypeptide moieties” as defined herein.

[0046] “Localization” includes determining the particular region of thesubject of an entity of interest, e.g., a tumor.

[0047] “Kemptide” includes a synthetic cAMP peptide substratecorresponding to part of the phosphyorylation site sequence in porcineliver pyruvate kinase. “Malantide” includes cAMP-dependent proteinkinase and protein kinase C substrate in various tissues.

[0048] “Small molecule” includes compositions that have a molecularweight of less than about 5 kD and most preferably less than about 4 kD.Small molecules can be, e.g., nucleic acids, peptides, polypeptides,peptidomimetics, carbohydrates, lipids or other organic or inorganicmolecules. “Spread of infection” includes the spreading and colonizationby a pathogen of host sites other than the initial infection site. Theterm can also include, however, growth in size and/or number of thepathogen at the initial infection site.

[0049] “Ligand” includes a molecule, a small molecule, a biomolecule, adrug, a peptide, a polypeptide, a protein, a protein complex, anantibody, a nucleic acid, or a cell.

[0050] “Ligand binding site” includes the location on thelight-generating fusion protein to which a ligand binds, whereupon thelight-generating polypeptide moiety is activated or inactivated as adirect or indirect consequence of ligand binding. Binding to the ligandbinding site may be direct or indirect, e.g., via protein dimerizationin conjunction with other proteins, as described hereinbelow.

[0051] “Targeting moiety” includes moieties which allow thelight-generating fusion protein of the invention to be selectivelydelivered to a target organ or organs. For example, if delivery of atherapeutic compound to the brain is desired, the carrier molecule mayinclude a moiety capable of targeting the compound to the brain, byeither active or passive transport. Illustratively, the carrier moleculemay include a redox moiety, as described in, for example, U.S. Pat. Nos.4,540,564 and 5,389,623, both to Bodor. These patents disclose drugslinked to dihydropyridine moieties which can enter the brain, where theyare oxidized to a charged pyridinium species which is trapped in thebrain. Thus, compound accumulates in the brain. Many targeting moietiesare known, and include, for example, asialoglycoproteins (see, e.g. Wu,U.S. Pat. No. 5,166,320) and other ligands which are transported intocells via receptor-mediated endocytosis. Targeting moieties may becovalently or non-covalently bound to a light-generating fusion protein.The targeting moiety may also be attached to a vector.

[0052] “Bioluminescent” molecules or moieties include luminescentsubstances which utilize chemical energy to produce light.

[0053] “Fluorescent” molecules or moieties include those which areluminescent via a single electronically excited state, which is of veryshort duration after removal of the source of radiation. The wavelengthof the emitted fluorescence light is longer than that of the excitingillumination (Stokes' Law), because part of the exciting light isconverted into heat by the fluorescent molecule.

[0054] “Entities” include, without limitation, small molecules such ascyclic organic molecules; macromolecules such as proteins; polymers;proteins; polysaccharides; nucleic acids; particles, inert materials;organelles; microorganisms such as viruses, bacteria, yeast and fungi;cells, e.g., eukaryotic cells; embryos; prions; tumors; all types ofpathogens and pathogenic substances; and particles such as beads andliposomes. In another aspect, entities may be all or some of the cellsthat constitute the mammalian subject being imaged, e.g., diseased ordamaged tissue, or compounds or molecules produced by those cells, or bya condition under study. Entities for which the invention has particularutility include tumors, proliferating cells, pathogens, and cellularenvironments comprising hypoxic tissue.

[0055] “Infectious agents” include parasites, viruses, fungi, bacteriaor prions.

[0056] “Promoter induction event” includes an event that results in thedirect or indirect induction of a selected inducible promoter.

[0057] “Heterologous gene” includes a gene which has been transfectedinto a host organism. Typically, a heterologous gene refers to a genethat is not originally derived from the transfected or transformedcells' genomic DNA.

[0058] “Opaque medium” includes a medium that is “traditionally” opaque,not necessarily absolutely opaque. Accordingly, an opaque mediumincludes a medium that is commonly considered to be neither transparentnor translucent, and includes items such as a wood board, and flesh andskin of a mammal.

[0059] “HIFα polypeptide moiety” includes all or part of the amino acidsequence of HIF1α, HIF2α, or HIF3α.

[0060] “HIF1α polypeptide moiety” includes all or part of the amino acidsequence of HIF1α, e.g., SEQ ID NO: 1X, the amino acid sequencecorresponding to the N-terminal residues 1-600 of HIF1α, numbered inaccordance with wild-type HIF1α, wherein either or both of residues 402and 564 are proline or hydroxylated proline, or an 80 to 120, 20 to 30,12 to 14, or 4 to 12 amino acid sequence corresponding to the residuesadjacent to and/or surrounding residue 402 and/or 564, inclusive, ofHIF1α, wherein residues 402 and/or 564 is proline or hydroxylatedproline.

[0061] The invention relates in part to methods and compositionsrelating to detecting, localizing and quantifying enzyme activities andprotein-protein interactions in vivo, in vitro and in silico usinglight-emitting fusion proteins. The fusion proteins contain domainscapable of binding by enzymes and other ligands, and of being modifiedas a consequence of this binding. The light generating domains include,without limitation, regions from fluorescent proteins and bioluminescentproteins. Light emission is detected by known methods, such as detectionwith suitable instrumentation (such as a CCD camera) in vivo, in vitroor in silico, such as in a living cell or intact organism, a cellculture system, a tissue section, or an array.

[0062] Light-generating fusion proteins of the invention are capable oftaking part in a luminescent reaction whereby different biological,biochemical, chemical and physical events are measured. Thelight-generating fusion protein is capable of being modified such thatit does or does not emit light or cause light to be emitted.Light-generating fusion proteins include a ligand binding site and alight-generating polypeptide moiety, wherein the bioluminescence of thepolypeptide moiety changes upon binding of a ligand at the ligandbinding site.

[0063] Without wishing to be bound by interpretation, the ligand bindingsite acts as in a sense as a “switch” for the light-generatingpolypeptide moiety, i.e., when the ligand binds to the ligand bindingsite, the light-generating polypeptide moiety emits light, oralternately, ceases to do so upon ligand binding. The “switching” on oroff, in embodiment, may be done by means of a “colinear effector site”which includes regions of the light-generating polypeptide moiety that,when acted on by events subsequent to ligand binding, cause thelight-generating polypeptide moiety to change its present light-emittingstate (i.e., on or off). The regions making up the colinear effectorsite may do this by, e.g., conformational distortion, chemicalmodification, e.g., ubiquitination of a residue or residues in thecolinear effector site, or by cleavage of a portion of all or part ofthe colinear effector site.

[0064] The invention further provides methods for testing putativeinhibitor compounds for activity (“screening”) in promoting HIFstabilization, e.g., contacting the compound, ischemic tissue, and thefusion protein of the invention under conditions appropriate to detectthe fusion protein if the compound promotes HIF stabilization. Themethod (also referred to herein as a “screening assay”) can be used foridentifying modulators, i.e., candidate or test compounds or agents(e.g., peptides, peptidomimetics, small molecules or other drugs) thatpromote HIF stabilization. The invention also includes compoundsidentified in the screening assays described herein, and pharmaceuticalcompositions for treatments as described herein.

[0065] Other screening methods are also part of the invention. Forexample, modulators of activity or latency of, or predisposition todisorders may be identified by administering a test compound to a testanimal at increased risk for a disorder (e.g., cancer, diabetes, heartdisease, stroke, or a hypoxia-related disorder), wherein the test animalrecombinantly expresses a light-generating fusion protein comprising aligand binding site and a light-generating polypeptide moiety, whereinthe light generation of the light-generating fusion protein changes uponbinding of a ligand at the ligand binding site, and the ligand bindingsite recognizes a ligand on an entity associated with a disorder, or aproduct of the disorder; allowing for localization of thelight-generating fusion protein and an entity, wherein contact betweenthe ligand binding site and a ligand associated with the disorder causesa modification of a colinear effector site which alters the lightgeneration of the light-generating polypeptide moiety; detecting theluminescence of the light-generating polypeptide moiety in the testanimal after administering the compound; and comparing the luminescenceof the light-generating polypeptide moiety in the test animal with theluminescence of the light-generating polypeptide moiety in a controlanimal, wherein a change in the activity of the light-generatingpolypeptide moiety in the test animal relative to the control indicatesthe test compound is a modulator of latency of or predisposition to, thedisorder in question.

[0066] The invention advantageously may be used to non-invasivelydetermine the effects of an anti-hypoxic compound in vivo. Alight-generating fusion protein of the invention, or a cell expressingsame, comprising an ubiquitin ligase binding site and a light-generatingpolypeptide moiety, wherein the light generation of the light-generatingfusion protein changes upon binding of a ubiquitin ligase at theubiquitin ligase binding site, the ubiquitin ligase binding siterecognizing a ubiquitin ligase present in hypoxic conditions in hypoxictissue is administered to a subject. Localization of thelight-generating fusion protein or cell in hypoxic tissue in the subject(wherein contact between the ubiquitin ligase binding site and aubiquitin ligase causes a modification of a colinear effector site whichalters the light generation of the light-generating polypeptide moiety)is allowed to occur, and the ability of the candidate compound toinhibit hypoxia is determined, by measuring the luminescence of thelocalized light-generating fusion protein.

[0067] The invention further relates to methods of identifying ordetecting prolyl hydroxylation, wherein the substrate peptide (orpolypeptide) is contacted with pVHL, wherein the amount of pVHL boundreflects the degree of hydroxylation. In one embodiment, the peptidecorresponds to HIF1α 555-575. The HIF peptide can be immobilized (forexample, on a nitrocellulose filter or at the bottom of a 96 well plate)or free in solution. Binding to pVHL can be monitored using a variety ofstandard methods familiar to those skilled in the art.

[0068] In another embodiment, the invention relates to methods ofidentifying or detecting prolyl hydroxylation, wherein the substratepeptide or polypeptide is contacted with an antibody, wherein the amountof antibody bound reflects the degree of hydroxylation. In oneembodiment, the peptide corresponds to HIF1α555-575. The HIF peptide maybe immobilized (for example, on a nitrocellulose filter or at the bottomof a 96 well plate) or free in solution. Binding to the antibody can bemonitored using a variety of standard methods familiar to those skilledin the art.

[0069] Yet another embodiment of the invention relates to methods ofidentifying or detecting prolyl hydroxylation wherein a polypeptide istranslated in the presence of labeled, e.g., radioactive, proline and aprolyl hydroxylase, hydrolyzing the resulting labeled polypeptide, anddetecting labeled hydroxyproline incorporation by analytical means, suchas thin layer chromatography.

[0070] A further embodiment of the invention relates to methods ofidentifying or detecting prolyl hydroxylation wherein the substratepeptide (or polypeptide) is contacted with a source of prolylhydroxylase in the presence or absence of putative inhibitors, and thedegree of prolyl hydroxylation is monitored as described in any one ofthe above three paragraphs. In one embodiment, the peptide correspondsto HIF1α 555-575, and the prolyl hydroxylase consists of a mammaliancell extract. In another embodiment, the peptide corresponds to HIF1α555-575 and the prolyl hydroxylase consists of purified or partiallypurified Egl9. Further, particularly useful embodiments relate to smallmolecule inhibitors of prolyl hydroxylation such as identified usingthis method, and use of the inhibitors to treat diseases characterizedby ischemia.

[0071] The test compounds of the invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds. See, e.g., Lam, 1997. AnticancerDrug Design 12: 145.

[0072] Libraries of chemical and/or biological mixtures, such as fungal,bacterial, or algal extracts, are known in the art and can be screenedwith any of the assays of the invention. Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb,et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, etal., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303;Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, etal., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,1994. J. Med. Chem. 37: 1233.

[0073] Libraries of compounds may be presented in solution (e.g.,Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991.Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556),bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat.No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; Ladner, U.S. Pat. No. 5,233,409.).

[0074] Modulation of prolyl hydroxylase has a variety of uses.Inhibiting prolyl hydroxylase may facilitate cell cycle progression andthe production of a number of proteins which promote angiogenesis and/orpromote cellular survival or cellular function in hypoxia, a desirableoutcome in the treatment of certain clinical conditions, particularlyischemic conditions such as coronary, cerebral and vascularinsufficiency.

[0075] VHL used in assays of the invention may be any suitable mammalianVHL, particularly human VHL. Human VHL has been cloned and sources ofthe gene can be readily identified by those of ordinary skill in theart. Its sequence is available as Genbank accession numbers AF010238 andL15409. Other mammalian VHLs are also available, such as murine VHL(accession number U 12570) and rat (accession numbers U 14746 andS80345). Non-mammalian homologues include the VHL-like protein of C.elegans, accession number F08G 12. VHL gene sequences may also beobtained by routine cloning techniques, for example by using all or partof the human VHL gene sequence as a probe to recover and to determinethe sequence of the VHL gene in other species. A wide variety oftechniques are available for this, for example, PCR amplification andcloning of the gene using a suitable source of mRNA (e.g., from anembryo or a liver cell), obtaining a cDNA library from a mammalian,vertebrate, invertebrate or fungal source, e.g., a cDNA library from oneof the above-mentioned sources, probing the library with apolynucleotide of the invention under stringent conditions, andrecovering a cDNA encoding all or part of the VHL protein of thatmammal. Suitable stringent conditions include hybridization on a solidsupport (filter) overnight incubation at 420° C. in a solutioncontaining 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate and 20 μg/ml salmon sperm DNA, followed by washing in 0.03Msodium chloride and 0.03M sodium citrate (i.e. 0.2×SSC) at from about50° C. to about 60° C.). Where a partial cDNA is obtained, the fulllength coding sequence may be determined by primer extension techniques.

[0076] It is not necessary to use the entire VHL protein (includingtheir mutants and other variants). Fragments of the VHL may be used,provided such fragments retain the ability to interact with the targetdomain of the HIFα subunit. Fragments of VHL may be generated in anysuitable way known to those of skill in the art. Suitable ways include,but are not limited to, recombinant expression of a fragment of the DNAencoding the VHL. Such fragments may be generated by taking DNA encodingthe VHL, identifying suitable restriction enzyme recognition siteseither side of the portion to be expressed, and cutting out that portionfrom the DNA. The portion may then be operably linked to a suitablepromoter in a standard commercially available expression system. Anotherrecombinant approach is to amplify the relevant portion of the DNA withsuitable PCR primers. Small fragments of the VHL (up to about 20 or 30amino acids) may also be generated using peptide synthesis methods whichare well known in the art. Generally fragments will be at least 40,preferably at least 50, 60, 70, 80 or 100 amino acids in size.

[0077] The HIFα subunit protein may be any human or other mammalianprotein, or fragment thereof which has the ability to bind to a wildtype full length VHL protein, such that the binding is able, in anormoxic cellular environment, to target the a subunit for destruction.

[0078] A number of HIFα subunit proteins have been cloned. These includeHIF1α, the sequence of which is available as Genbank accession numberU22431, HIF2α, available as Genbank accession number U81984 and HIF3α,available as Genbank accession numbers AC007193 and AC079154. These areall human HIFα subunit proteins. HIFα subunit proteins from otherspecies, including murine HIF1α (accession numbers AF003695, US9496 andX95580), rat HIF1α (accession number Y09507), murine HIF2α (accessionnumbers U81983 and D89787) and murine HIF3α (accession number AF060194).Other mammalian, vertebrate, invertebrate or fungal homologues may beobtained by techniques similar to those described above for obtainingVHL homologues.

[0079] Variants of the HIFα subunits may be used, such as syntheticvariants which have at least 45% amino acid identity to a naturallyoccurring HIFα subunit (particularly a human HIFα subunit), preferablyat least 50%, 60%, 70%, 80%, 90%, 95% or 98% identity.

[0080] Fragments of the HIFα subunit protein and its variants may beused, provided that the fragments retain the ability to interact with awild-type VHL, preferably wild-type human VHL. Such fragments aredesirably at least 20, preferably at least 40, 50, 75, 100, 200, 250 or400 amino acids in size. Alternately, such fragments may be 12 to 14amino acids in size, or as small as four amino acids. Most desirablysuch fragments include the region 555-575 found in human HIF1α or itsequivalent regions in other HIFα subunit proteins. Optionally thefragments also include one or more domains of the protein responsiblefor transactivation. Reference herein to a HIFα subunit protein includesthe above mentioned mutants and fragments which are functionally able tobind VHL protein unless the context is explicitly to the contrary.

[0081] The percentage homology (also referred to as identity) of DNA andamino acid sequences can be calculated using commercially availablealgorithms. The following programs (provided by the National Center forBiotechnology Information) may be used to determine homologies: BLAST,gapped BLAST and PSI-BLAST, which may be used with default parameters.The algorithm GAP (Genetics Computer Group, Madison, Wis.) uses theNeedleman and Wunsch algorithm to align two complete sequences thatmaximizes the number of matches and minimizes the number of gaps.Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of either of the terms“homology” and “homologous” herein does not imply any necessaryevolutionary relationship between compared sequences, in keeping forexample with standard use of terms such as “homologous recombination”which merely requires that two nucleotide sequences re sufficientlysimilar to recombine under the appropriate conditions.

[0082] The precise format of the screening assays may be varied usingroutine skill and knowledge. The amount of VHL, HIFα subunit and, whererequired, further components, may be varied depending upon the scale ofthe assay. In general, the person of skill in the art will selectrelatively equimolar amounts of the two components, say from 1:10 to100:1, preferably from 1:1 to 10:1. molar ratio of VHL to HIFα subunit.However there may be particular assay formats which can be practicedoutside this range.

[0083] Where assays of the invention are performed within cells, thecells may be treated to provide or enhance a normoxic environment. By“normoxic” it is meant levels of oxygen similar to those found in normalair, e.g. about 21% O₂ and 5% CO₂, the balance being nitrogen. Ofcourse, these exact proportions do not have to be used, and may bevaried independently of each other. Generally a range of from 10-30%oxygen, 1-10% CO₂ and a balance of nitrogen or other relatively inertand non-toxic gas may be used. Normoxia may be induced or enhanced incells, for example by culturing the cells in the presence of hydrogenperoxide as described above.

[0084] Alternatively, or by way of controls, cells may also be culturedunder hypoxic conditions. By “hypoxic” it is meant an environment withreduced levels of oxygen. Most preferably oxygen levels in cell culturewill be 0.1 to 1.0% for the provision of a hypoxic state. Hypoxia may beinduced in cells simply by culturing the cells in the presence oflowered oxygen levels. The cells may also be treated with compoundswhich mimic hypoxia and cause up regulation of HIFα subunit expression.Such compounds include iron chelators, cobalt (II), nickel (II) ormanganese (II), all of which may be used at a concentration of 20 to 500μM. such as 100 μM. Iron chelators include desferrioxamine,O-phenanthroline or hydroxypyridinones (e.g. 1,2-diethylhydroxypyridinone (CP94) or 1,2-dimethyl hydroxypyridinone (CP20)).

[0085] Cells in which assays of the invention may be preformed includeeukaryotic cells, such as yeast, insect, mammalian, primate and humancells. Mammalian cells may be primary cells or transformed cells,including tumor cell lines. The cells may be modified to express or notto express other proteins which are known to interact with HIF (xsubunit proteins and VHL protein, for example Elongin C and Elongin Bproteins in the case of VHL and ARNT protein, in the case of HIFαsubunit protein.)

[0086] In cell free systems such additional proteins may be included,for example by being provided by expression from suitable recombinantexpression vectors.

[0087] In assays performed in cells, it will be desirable to achievesufficient expression of VHL to recruit sufficient HIFα subunit to acomplex such that the effect of a putative modulator compound may bemeasured. The level of expression of VHL and HIFα subunit may be variedwithin fairly wide limits, so that the intracellular levels of the twomay vary by a wide ratio, for example from 1:10 to 1000:1, preferably1:1 to 100:1, molar ratio of VHL to HIFα subunit.

[0088] The amount of putative modulator compound which may be added toan assay of the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 μM concentrations of putative modulator compound may be used, forexample from 0.1 to 10 μM. Modulator compounds may be those which eitheragonize or antagonize the interaction. Antagonists (inhibitors) of theinteraction are particularly desirable.

[0089] Modulator compounds which may be used may be natural or syntheticchemical compounds used in drug screening programs. Extracts of plantswhich contain several characterized or uncharacterized components mayalso be used.

[0090] The invention provides methods for determining hypoxicconditions, cancer or infection in an individual to thereby selectappropriate therapeutic or prophylactic agents for that individual(referred to herein as “pharmacogenomics”). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual examined to determine the abilityof the individual to respond to a particular agent.) Yet another aspectof the invention pertains to monitoring the influence of agents (e.g.,drugs, compounds) on hypoxic conditions, cancer or infection in clinicaltrials.

[0091] Thus, the diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder associated with hypoxic conditions, cancer or infection.Furthermore, the prognostic assays described herein can be used todetermine whether a subject should be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with hypoxic conditions, cancer or infection.

[0092] The selection of a light-generating polypeptide moiety of thelight-generating fusion protein should be done so as to produce lightcapable of penetrating animal tissue such that it can be detectedexternally in a non-invasive manner. The ability of light to passthrough a medium such as animal tissue (composed mostly of water) isdetermined primarily by the light's intensity and wavelength.

[0093] The more intense the light produced in a unit volume, the easierthe light will be to detect. The intensity of light produced in a unitvolume depends on the spectral characteristics of individuallight-generating polypeptide moieties, and on the concentration of thosemoieties in the unit volume. Accordingly, schemes that place a highconcentration of light-generating polypeptide moieties in or on anentity (such as high-efficiency loading of a liposome or high-levelexpression of a light-generating fusion protein in a cell) typicallyproduce brighter light-generating fusion proteins (LGPs), which areeasier to detect through deeper layers of tissue, than schemes whichconjugate, for example, only a single LGM onto each entity.

[0094] A second factor governing detectability through a layer of tissueis the wavelength of the emitted light. Water may be used to approximatethe absorption characteristics of animal tissue, since most tissues arecomposed primarily of water. It is well known that water transmitslonger-wavelength light (in the red range) more readily than it doesshorter wavelength light.

[0095] Accordingly, light-generating polypeptide moieties which emitlight in the range of yellow to red (550-1100 nm) are typicallypreferable to those which emit at shorter wavelengths. However,excellent results can be achieved in practicing the present inventionwith LGMs that emit in the range of 486 nm, despite the fact that thisis not an optimal emission wavelength.

[0096] Fluorescence-based Moieties. Because fluorescent moleculesrequire input of light in order to luminesce, their use in the inventionmay be more involved than the use of bioluminescent molecules.Precautions are typically taken to shield the excitatory light so as notto contaminate the fluorescence photon signal being detected from thesubject. Obvious precautions include the placement of an excitationfilter at the radiation source. An appropriately-selected excitationfilter blocks the majority of photons having a wavelength similar tothat of the photons emitted by the fluorescent moiety. Similarly, abarrier filter is employed at the detector to screen out most of thephotons having wavelengths other than that of the fluorescence photons.Filters such as those described above can be obtained from a variety ofcommercial sources, including Omega Optical, Inc. (Brattleboro, Vt.).

[0097] Alternatively, a laser producing high intensity light near theappropriate excitation wavelength, but not near the fluorescenceemission wavelength, can be used to excite the fluorescent moieties. Anx-y translation mechanism may be employed so that the laser can scan thesubject, for example, as in a confocal microscope.

[0098] As an additional precaution, the radiation source may be placedbehind the subject and shielded, such that the only radiation photonsreaching the site of the detector are those that pass all the waythrough the subject. Furthermore, detectors may be selected that have areduced sensitivity to wavelengths of light used to excite thefluorescent moiety.

[0099] An advantage of small fluorescent molecules is that they are lesslikely to interfere with the bioactivity of the entity to which they areattached than a would a larger light-generating moiety. In addition,commercially-available fluorescent molecules can be obtained with avariety of excitation and emission spectra that are suitable for usewith the present invention. For example, Molecular Probes (Eugene,Oreg.) sells a number of fluorophores, including Lucifer Yellow (abs. at428 nm, and emits at 535 nm) and Nile Red (abs. at 551 nm and emits at636 nm). Further, the molecules can be obtained derivatized with avariety of groups for use with various conjugation schemes (e.g., fromMolecular Probes).

[0100] Bioluminescence-based Moieties. The subjects of chemiluminescence(luminescence as a result of a chemical reaction) and bioluminescence(visible luminescence from living organisms) have, in many aspects, beenthoroughly studied.

[0101] An advantage of bioluminescent moieties over fluorescentmoieties, is that there is virtually no background in the signal. Theonly light detected is light that is produced by the exogenousbioluminescent moiety. In contrast, the light used to excite afluorescent molecule often results in the fluorescence of substancesother than the intended target. This is particularly true when the“background” is as complex as the internal environment of a livinganimal.

[0102] Ligands include, in a preferred embodiment, enzymes which arecapable of modifying a light-generating polypeptide moiety such that itdoes or ceases to emit light when modified. In use, this happens when,e.g., the light-generating fusion protein comes in contact with a ligandon an entity or produced by the entity, and the ligand binds to theligand binding site, altering the light-generating properties of thelight-generating polypeptide moiety. Examples include the following.

[0103] In an especially useful embodiment of the present invention, alight-generating fusion protein comprising a binding site for an E3ubiquitin ligase and a light-generating polypeptide moiety capable ofbeing modified by E3 at a modification site can be used for diagnosticand treatment purposes. Ubiquitin ligases (e.g., E3) bind to colinear‘T’ (see FIG. 1) present on their substrates. Examples include the SCF(Skp1/Cdc53/F-box) ubiquitin ligases, the VBC (pVHL/elongin B/elongin C)ubiquitin ligase, and the MDM2 ubiquitin ligase. It is alreadyestablished that light-generating proteins such as GFP and luciferasecan be fused to heterologous polypeptides without loss of activity. Insome embodiments, the light-generating polypeptide moiety may bemodified to include a surface exposed lysine residue accessible as aubiquitin acceptor site. In a First embodiment, a light-generatingfusion protein of the invention may be used to monitor ischemia inliving tissues and animals. The First embodiment comprises a polypeptidederived from the HIF1α (hypoxia-inducible factor 1α) which acts as abinding site for VBC. This binding site is only recognized by VBC in thepresence of oxygen as a result of proline hydroxylation. Thelight-generating fusion protein of the First embodiment isadvantageously stable in hypoxic cells, but unstable in well oxygenatedcells. In a related embodiment, the HIF-derived polypeptide describedabove may be fused to a suicide moiety such as a protein (such as HSV TKor an adenoviral protein like E1A), which can be used in selectivekilling of ischemic cells (such as in a solid tumor).

[0104] Binding and/or modification sites are well-known in the art for avariety of kinases including cyclin-dependent kinases, ATM/ATR, JNKkinases, and receptor tyrosine kinases. In one embodiment, alight-generating fusion protein may be fused to a binding site for akinase of interest. In some embodiments, it is useful to introduce oneor more binding sites for a selected kinase into the light-generatingfusion protein. By way of example, since phosphorylated serine,threonine, and tyrosine, by virtue of their negative charges, frequentlymimic aspartic acid and/or glutamic acid residues, individual asparticacid and glutamic acid residues are replaced with serine, threonine, ortyrosine (in the context of the kinase modification site). Suchsubstitutions may be made singly and in combination. In anotherembodiment, the modification site can be empirically determined bycarrying out a linker scan of the light-generating protein using alinker encoding the kinase modification site. In yet another embodimentthe light-generating polypeptide moiety is mutagenized (either random ortargeted mutagenesis) to generate modification sites in which thelight-generating polypeptide moiety is selectively inactivated oractivated by the kinase; this mutagenesis is performed using cells (suchas yeast) rendered conditional for the kinase. In still yet anotherembodiment, a kinase modification site is designed in silico based oncomparison of the binding site of the selected kinase to the primarysequence of the light-generating polypeptide moiety, coupled withknowledge of the three dimensional structure of the light-generatingpolypeptide moiety.

[0105] In an additional embodiment described below, a light-generatingpolypeptide moiety is fused to a polypeptide recognized by a cyclin/cdk2complex. This enzyme phosphorylates serine or threonine with an absoluterequirement for proline in the +1 position. By way of example, GFPcontains 13 proline residues including one threonine-proline site andtwo aspartic acid-proline sites. In one embodiment, a light-generatingprotein (e.g., GFP) is fused to a cyclin/cdk2 binding site such asY-Lys-X₁-Leu-K-X₂-Leu-Y′ (SEQ ID NO. 9). The light-generating protein isphosphorylated and inactivated by cyclin/cdk2, providing a detectablesignal which is selectively off in the presence of cyclin/cdk2. Inanother embodiment, the light-generating polypeptide moiety is mutatedso that it is not phosphorylated and activated by cyclin/cdk2. Anexample of this mutation would be to mutate the two asparticacid-proline sites to serine (or threonine)-prolines.

[0106] In this additional embodiment, the ligand binding site is apolypeptide recognized by a cyclin/cdk2 complex, e.g., comprising theamino acid sequence Y-Lys-X₁-Leu-K-X₂-Leu-Y′, wherein X₁, X₂, areindependently any one or more amino acids; and Y and Y′ areindependently present or absent and, if present, independently comprisea peptide having from 1 to 600 amino acids.

[0107] An example of such a target peptide is RB: PKPLKKLRFD (SEQ ID NO:1). In a related aspect of this additional embodiment, the ligandbinding site comprises the amino acid sequence Y-X₁-Arg-Arg-Leu-Y′,wherein X₁ is Lys or Cys; and Y and Y′ are independently present orabsent and, if present, independently comprise a peptide having from 1to 600 amino acids. Two non-limiting examples of the target peptide ofthis related aspect of this additional embodiment are: E2F1:GRPPVKRRLDLE (SEQ ID NO: 2); derived from the E2F1 protein, and p21:CCSKACRRLFGP (SEQ ID NO: 3), derived from the p21 protein.

[0108] The fusion protein of this additional embodiment can be producedby standard recombinant DNA techniques, discussed supra.

[0109] The ligand binding site of the light-generating fusion protein ofthis additional embodiment is derived from the retinoblastoma (RB)protein. Adams, et al., 1999 Mol. Cell Biol. 19:1068 describes RBprotein. An RB polypeptide comprises 928 amino acid residues. Asdescribed in more detail below, this ligand binding site comprises aunique cyclin binding domain derived from RB. In the light-generatingfusion protein described above, where present, Y comprises between 1 and900 amino acid residues, preferably corresponding to the sequence of“N-terminal” RB amino acid residues 1-868. Thus, in a preferredembodiment Y can represent any sequence of N-terminal amino acids of RB,for example residues 794-829, and so on up to and including residues1-868. Y′ can also comprise between 1 and 600 amino acid residues,preferably corresponding to the sequence of “C-terminal” RB amino acidresidues 879-928. Thus, in a preferred embodiment, Y′ can represent anysequence of C-terminal amino acids of RB, for example 879-910, and so onup to and including residues 879-928. Y and Y′ can also containconservative substitutions of amino acids present in the N-terminal andC-terminal RB sequences described above. In a further preferredembodiment, Y and Y′ are absent.

[0110] The light-generating fusion protein of this additional embodimentprovides a way for detecting “cancerous tissue” or tissue subject toaberrant cell proliferation and therefore at risk for cancer. Inaddition to tissue that becomes cancerous due to an in situ neoplasm,for example, the light-generating fusion protein also provides a methodof detecting cancerous metastatic tissue present in distal organs and/ortissues. Thus such tissue may be detected by contacting tissue suspectedof being cancerous with the light-generating fusion protein underappropriate conditions to cause the phosphorylation of thelight-generating fusion protein in cancerous tissue, thereby detectingthe presence of cancerous tissue. The ligand binding site of thelight-generating fusion protein of this embodiment provides a bindingsite for cyclin-cyclin-dependent kinase (cdk) complexes whichphosphorylate the protein, causing it to emit or not emit light in thepresence of active cycle-cdk complexes. Under appropriate conditions,the light-generating fusion protein will therefore be phosphorylated orinactive in tissue that is not cancerous, and unphosphorylated andpreserving its light-emitting properties in tissue that is cancerous,e.g. primary and metastatic tissue.

[0111] In another useful embodiment, a light-generating polypeptidemoiety is fused to a polypeptide recognized by a cyclin/cdk2 complex.This enzyme phosphorylates serine or threonine with an absoluterequirement for proline in the +1 position. By way of example, GFPcontains 13 proline residues including one threonine-proline site andtwo aspartic acid-proline sites. In one embodiment, a light-generatingprotein (e.g., GFP) is fused to a cyclin/cdk2 binding site such asY-Lys-X₁-Leu-K-X₂-Leu-Y′ (SEQ ID NO. 9). The light-generating protein isphosphorylated and inactivated by cyclin/cdk2, providing a detectablesignal which is selectively off in the presence of cyclin/cdk2. Inanother embodiment, the light-generating polypeptide moiety is mutatedso that it is not phosphorylated and activated by cyclin/cdk2. Anexample of this mutation would be to mutate the two asparticacid-proline sites to serine (or threonine)-prolines.

[0112] Phosphorylation is one of the most important ways toposttranslationally modify proteins, and it regulates diverse cellphysiological processes (transport, proliferation, differentiation). Forexample, phosphorylation is involved in all phases of cell division: intransition from G1 to S phase, progression of cells during S phase andentry into M phase. The physiological function of oncoproteins and tumorsuppressor proteins that are involved in gene expression and replicationare also regulated by phosphorylation. Many growth factors and theirreceptors are encoded by oncogenes which are mutated or overexpressed ina variety of human tumors. Mutation or overexpression of these oncogenesleads to unchecked cell division, and transformation of normal cells tomalignant. In an embodiment of the present invention, a light-generatingfusion protein may include a phosphatase binding site and a modificationsite, such as a phosphorylated amino acid residue capable of beingmodified by a protein “phosphatase”.

[0113] For most proteases, the enzyme binding site and modification siteare largely congruent and can be encompassed in short peptides. In anembodiment, a light-generating fusion protein may include a proteasebinding site and a modification site capable of being cleaved by theprotease. The binding site and modification site may be the same site orin two discrete regions. In one embodiment, wherein the binding andmodification site are congruent, linker scanning mutagenesis of a givenlight-generating protein is carried out using a linker that encodes thecongruent binding/modification site. Resulting mutants are thoselight-generating fusion proteins containing a binding/modification sitethat preserves light-emitting activity in protease deficient cells andexhibits protease sensitivity in vitro and in vivo. In the Thirdembodiment described herein, a light-generating fusion proteincontaining an HIV protease site is especially useful for monitoring thepresence or absence of HIV (in this case, with HIV-positive cells notemitting light). In an alternative embodiment, the light-generatingfusion protein is fused via a linker to a protein that inhibits thelight-emitting activity of the light-generating polypeptide moiety. Thelinker includes a binding/modification site for an HIV protease. Thus,cleavage at the binding/modification site should remove the inhibitingprotein moiety, thus yielding a positive signal in cells that are HIVpositive.

[0114] As shown generally in FIG. 1A, an embodiment of the fusionprotein of the invention contains a ligand binding site “T”, a reporterdomain (e.g., light-generating polypeptide moiety) “R”, and amodification site “X”. Binding of enzyme “E” (the ligand) to target site“T” results in a modification of modification site “X”, which causes thereporter domain “R” to either emit or not emit light.

[0115] The site “T” and modification site “X” may be separate and in cison the fusion protein, as shown in FIG. 1B. Either “T” or “X” can beproximal or distal to the amino terminus of the fusion protein.

[0116] In another embodiment shown in FIG. 1C, wherein the “T” and the“X” of the fusion protein are congruent within a domain of the fusionprotein. In related embodiments, the congruent “T/X” can be at the aminoterminus, the carboxy terminus, or at neither terminus of the fusionprotein.

[0117] In a further embodiment shown in FIG. 1D, the “T” is on a proteinassociated with the fusion protein containing the “X”. In a relatedembodiment, target site “T” is on the fusion protein and modificationsite “X” is on a protein that is physically associated with the fusionprotein, wherein modification of the associated protein results in thefusion protein either emitting or not emitting light. The depiction ofsite “T” and modification site “X” are not intended to be limiting. “T”can be at the amino terminus, the carboxy terminus, or at neitherterminus of the fusion protein, and “X” can be at the amino terminus,the carboxy terminus, or at neither terminus of the associated protein.

[0118] The invention also includes a fusion protein in which the ligandbinding site sequence “T” is unknown. In this embodiment, e.g., as shownin FIG. 2A, the fusion protein includes a first hetero- orhomo-dimerization domain (“HD1”). An enzyme “E” capable of modifyingmodification site “X” on the fusion protein is fused to a second hetero-or homo-dimerization domain “HD2” that interacts with “HD1”. In arelated embodiment, the targeting site “T” for an enzyme “E” can begenerated by inserting one or more polypeptide sequences derived from arandom or non-random peptide library into the fusion protein.

[0119] In a related embodiment (FIG. 2B), a binding domain of a protein“A” containing an enzyme target site “T” interacts with a binding domain“B” of a fusion protein, which results in enzyme “E” modifying thereporter domain “R” of fusion protein “B” at modification site “X” suchthat the fusion protein does or does not emit light or cause light to beemitted. In the First embodiment, the ligand binding site comprises apolypeptide derived from the HIF1α (hypoxia-inducible factor 1α) whichacts as a binding site for VBC, e.g., the amino acid sequenceY-X₁-Leu-X₂-Pro_(h)-X₃-X₄-X₅-X₆-Y′, wherein

[0120] Pro_(h) is hydroxylated proline;

[0121] X₁, X₂, X₃, X₄, X₅, and X₆ are amino acids selected so as to notmodify or alter VHL binding properties. X₁, X₂, X₄, X₅, and X₆ aredesirably independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp,and X₃ is desirably Ser, Thr, or Tyr; and

[0122] Y and Y′ are independently present or absent and, if present,independently comprise a peptide having from 1 to 600 amino acids.

[0123] In a preferred embodiment, the ligand binding site comprises theamino acid sequence corresponding to the N-terminal residues 1-600 ofHIF1α, wherein either or both of residues 402 and 564 are proline orhydroxylated proline. In a more preferred embodiment, the ligand bindingsite comprises an 80 to 120, 20 to 30, 12 to 14, or 4 to 12 amino acidsequence corresponding to the residues adjacent to and/or surroundingresidue 402 and/or 564, inclusive, of HIF1α, wherein residues 402 and/or564 is proline or hydroxylated proline. “Residues adjacent to and/orsurrounding” is meant to include the relevant sequence of HIF1α eitherbefore, after, or flanking, the specified residue, e.g., residue 564.

[0124] Such proteins may be used effectively as oxygen-sensing proteins.The fusion proteins may be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques, e.g., by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, 1992).

[0125] The ligand binding site noted above is derived from HIF1αprotein. U.S. Pat. No. 6,222,018, describes HIF protein and itspreparation in substantially pure form. HIF is composed of subunitsHIF1α and an isoform HIF1β. HIF1α polypeptide comprises 826 amino acidresidues. As described in more detail below, the ligand binding sites ofthis particular fusion protein comprises a unique ubiquitin ligasebinding domain derived from HIF1α. In this fusion protein, wherepresent, Y comprises between 1 and 600 amino acid residue, preferablycorresponding to the sequence of “N-terminal” HIF1α amino acid residues1-555. Thus, in a preferred embodiment Y can represent any sequence ofN-terminal amino acids of HIF1α, for example residues 554-555, 553-555,552-555, and so on up to and including residues 1-555. Y′ can alsocomprises between 1 and 600 amino acid residues, preferablycorresponding to the sequence of “C-terminal” HIF1α amino acid residues576-826. Thus, in a preferred embodiment, Y′ can represent any sequenceof C-terminal amino acids of HIF1α, for example 576-577, 576-578,576-579, and so on up to and including residues 576-826. Y and Y′ canalso contain conservative substitutions of amino acids present in theN-terminal and C-terminal HIF1α sequences described above. In apreferred embodiment of the ligand binding site defined above, Y and Y′are absent. In a further preferred embodiment, X₁ is Met, X₂ is Leu, X₃is Ala, X₄ is Tyr, X₅ is Pro, and X₆ is Met. In a particularly preferredembodiment of the fusion protein, the ligand binding site has the aminoacid sequenceAsp-Leu-Asp-Leu-Glu-Met-Leu-Ala-Proh-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu-Arg,corresponding to HIF1α amino acid residues 556-575, with a hydroxylatedproline at amino acid residue 564.

[0126] The invention also provides a nucleic acid molecule encoding thefusion protein or polypeptide of the invention. (As used herein, theterms polypeptide and protein are interchangeable). An “isolated”nucleic acid molecule is one that is separated from other nucleic acidmolecules that are present in the natural source of the nucleic acid.Examples of isolated nucleic acid molecules include, but are not limitedto, recombinant DNA molecules contained in a vector, recombinant DNAmolecules maintained in a heterologous host cell, partially orsubstantially purified nucleic acid molecules, and synthetic DNA or RNAmolecules. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

[0127] A nucleic acid of the invention can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to the nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer. The present invention therefore also providesa vector comprising the nucleic acid of the invention. In a preferredembodiment, the vector further comprises a promoter operably linked tothe nucleic acid molecule. In a further preferred embodiment theinvention provides a cell containing the vector of the invention.

[0128] The light-generating fusion protein of, e.g., the Firstembodiment provides a means for detecting hypoxic tissue or tissuesubject to chronic hypoxia and therefore at risk for ischemia. Inaddition to tissue that becomes ischemic due to occurrence of a stroke,heart attack or embolism, for example, the fusion protein also providesa method of detecting ischemic tissue present in tumors. Thus theinvention may be used to detect such tissue by contacting tissuesuspected of being hypoxic with the light-generating fusion proteinunder appropriate conditions to cause the ubiquitination of the fusionprotein in normoxic tissue, thereby detecting the presence of hypoxictissue. As described more fully below, the ligand binding site of thelight-generating fusion protein also provides a binding site forubiquitin ligases which destroy the protein in the presence of oxygen.Under appropriate conditions, the light-generating fusion protein of theinvention will therefore be “unstable” or destroyed in tissue that isnot hypoxic, i.e., that is well oxygenated, and “stable” or preservingits light-emitting properties in tissue that is hypoxic, i.e. that lackssufficient oxygen.

[0129] The Third embodiment provides a means for detecting “infectedtissue” or tissue subject to contact with infectious agents andtherefore at risk for infection, e.g., monitoring the presence orabsence of HIV. The ligand binding site of the Third embodimentcomprises a binding site for a protease, such as an infection-associatedprotease. For example, protease cleavage sites of the humanimmunodeficiency virus (HIV-1) protein precursor Pr55 (gag) proteininclude p2/NC, NC/p1, and NC/TFP.

[0130] The ligand binding site of the light-generating fusion protein ofthis Third embodiment may be derived from the HIV-1 protein. Onenon-limiting example of the target peptide of this Third embodiment is:

[0131] Y-GSGIF*LETSL-Y′ (See Beck et al., (2000) Virology274(2):391-401.

[0132] Y and Y′ are independently present or absent and, if present,independently comprise a peptide having from 1 to 600 amino acids, and“*” indicates the cleavage site of the fusion protein by a protease.

[0133] In addition to tissue that becomes infected due to an acute orchronic infection by an infectious agent, for example, thelight-generating fusion protein may be used in detecting infected tissuepresent in distal organs and/or tissues, e.g., by contacting tissuesuspected of being infected with the light-generating fusion protein ofthe invention under appropriate conditions to cause the proteolysis ofthe light-generating fusion protein in infected tissue, therebydetecting the presence of infected tissue. The ligand binding site ofthe light-generating fusion protein of this Third embodiment provides abinding site for a protease which degrades or modifies the protein,causing it to emit or not emit light in the presence of one or moreproteases. Under appropriate conditions, the light-generating fusionprotein will therefore be proteolyzed or inactive in tissue that isinfected (although there may be cases where proteolysis leads to lightgeneration), and unproteolyzed and preserving its light-emittingproperties in tissue that is infected.

[0134] A Fourth embodiment of a light-generating fusion protein of theinvention comprises a light-generating protein moiety and a ligandbinding site, wherein the ligand binding site comprises an amino acidsequence capable of binding to an “associated” protein. The associationcan occur by covalent or non-covalent binding. This associated proteinmay itself be a light-generating fusion protein, comprising a bindingpolypeptide capable of binding to the light-emitting light-generatingfusion protein and an enzyme capable of modifying the light-emittinglight-generating fusion protein.

[0135] A non-limiting example of the target peptide of this Fourthembodiment is:

[0136] HD1: WFHGKLSR (Amino acids 488-495 of Accession No. P29353, humanSHC1; SEQ ID NO: 4).

[0137] This target polypeptide contains an SH2 domain. Therefore, anyassociated protein with an SH2 domain should interact with the targetpeptide of the Fourth embodiment. A non-limiting example of the bindingpeptide of the associated protein of this Fourth embodiment is:

[0138] HD2: WNVGSSNR (Amino acids 624-631 of Accession No. P27986; humanP13K p85 subunit; SEQ ID NO: 5).

[0139] Another non-limiting example of the Fourth embodiment would be tofuse the FK506 binding protein (FKBP12) domain moiety to GFP and theFRAP domain moiety to Skp1 or elonginC. Therefore in the presence ofrapamycin, which promotes the high affinity interaction of FKBP12 andFRAP, the core E3 ligase machinery would bind to and destroy GFP,eliminating the bioluminescence wherever rapamycin is present.

[0140] The ligand binding site of the light-generating fusion protein ofthis Fourth embodiment is derived from the human SHC1 proteinprotein.See Pelicci et al., (1992) Cell 70:93-104, describes the SHC1 proteinwith an SH2 domain which is implicated in mitogenic signal transduction.

[0141] The light-generating fusion protein of this Fourth embodimentprovides a means for detecting enzymatic activity where the enzymebinding site is undefined.

[0142] The pharmaceutical compositions of the invention comprise thenovel agents combined with a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0143] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0144] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0145] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0146] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0147] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0148] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0149] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0150] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0151] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0152] The invention includes entities which may have been modified orconjugated to include a light-generating fusion protein of theinvention. Such conjugated or modified entities are referred to aslight-emitting entities, or simply conjugates. The conjugates themselvesmay take the form of, for example, molecules, macromolecules, particles,microorganisms, or cells. The methods used to conjugate alight-generating fusion protein to an entity depend on the nature of thelight-generating fusion protein and the entity. Exemplary conjugationmethods are discussed in the context of the entities described below.

[0153] Small molecules. Small molecule entities which may be useful inthe present invention include compounds which specifically interact witha pathogen or an endogenous ligand or receptor. Examples of suchmolecules include, but are not limited to, drugs or therapeuticcompounds; toxins, such as those present in the venoms of poisonousorganisms, including certain species of spiders, snakes, scorpions,dinoflagellates, marine snails and bacteria; growth factors, such asNGF, PDGF, TGF and TNF; cytokines; and bioactive peptides.

[0154] The small molecules are preferably conjugated to light-generatingfusion proteins in a way that that the bioactivity of the small moleculeis not substantially affected. Conjugations are typically chemical innature, and can be performed by any of a variety of methods known tothose skilled in the art.

[0155] Small molecules conjugated to light-generating fusion proteins ofthe present invention may be used either in animal models of humanconditions or diseases, or directly in human subjects to be treated. Forexample, a small molecule which binds with high affinity to receptorexpressed on tumor cells may be used in an animal model to localize andobtain size estimates of tumors, and to monitor changes in tumor growthor metastasis following treatment with a putative therapeutic agent.Such molecules may also be used to monitor tumor characteristics, asdescribed above, in cancer patients.

[0156] Macromolecules. Macromolecules, such as polymers and biopolymers,constitute another example of entities useful in practicing the presentinvention. Exemplary macromolecules include antibodies, antibodyfragments, light-generating fusion proteins and certain vectorconstructs.

[0157] Antibodies or antibody fragments, purchased from commercialsources or made by methods known in the art, can be used to localizetheir antigen in a mammalian subject by conjugating the antibodies to alight-generating polypeptide moiety, administering the conjugate to asubject by, for example, injection, allowing the conjugate to localizeto the site of the antigen, and imaging the conjugate.

[0158] Antibodies and antibody fragments have several advantages for useas entities in the present invention. By their nature, they constitutetheir own targeting moieties. Further, their size makes them amenable toconjugation with several types of light-generating fusion proteins,including small fluorescent molecules and fluorescent and bioluminescentproteins, yet allows them to diffuse rapidly relative to, for example,cells or liposomes.

[0159] The light-generating fusion proteins can be conjugated directlyto the antibodies or fragments, or indirectly by using, for example, afluorescent secondary antibody. Direct conjugation can be accomplishedby standard chemical coupling of, for example, a fluorophore to theantibody or antibody fragment, or through genetic engineering. Chimeras,or fusion proteins can be constructed which contain an antibody orantibody fragment coupled to a fluorescent or bioluminescent protein.For example, Casadei, et al., describe a method of making a vectorconstruct capable of expressing a fusion protein of aequorin and anantibody gene in mammalian cells.

[0160] Conjugates containing antibodies can be used in a number ofapplications of the present invention. For example, a labeled antibodydirected against E-selection, which is expressed at sites ofinflammation, can be used to localize the inflammation and to monitorthe effects of putative anti-inflammatory agents.

[0161] Vector constructs by themselves can also constitutemacromolecular entities applicable to the present invention. Forexample, a eukaryotic expression vector can be constructed whichcontains a therapeutic gene and a gene encoding a light-generatingmolecule under the control of a selected promoter (i.e., a promoterwhich is expressed in the cells targeted by the therapeutic gene).Expression of the light-generating molecule, assayed using methods ofthe present invention, can be used to determine the location and levelof expression of the therapeutic gene. This approach may be particularlyuseful in cases where the expression of the therapeutic gene has noimmediate phenotype in the treated individual or animal model.

[0162] Viruses. Another entity useful for certain aspects of theinvention are viruses. As many viruses are pathogens which infectmammalian hosts, the viruses may be conjugated to a light-generatingfusion protein and used to study the initial site and spread ofinfection. In addition, viruses labeled with a light-generating fusionprotein may be used to screen for drugs which inhibit the infection orthe spread of infection.

[0163] A virus may be labeled indirectly, either with an antibodyconjugated to a light-generating fusion protein, or by, for example,biotinylating virions as known in the art and then exposing them tostreptavidin linked to a detectable moiety, such as a fluorescentmolecule.

[0164] Alternatively, virions may be labeled directly with a fluorophorelike rhodamine, using methods known in the art. The virus can also begenetically engineered to express a light-generating fusion protein.Labeled virus can be used in animal models to localize and monitor theprogression of infection, as well as to screen for drugs effective toinhibit the spread of infection. For example, while herpes virusinfections are manifested as skin lesions, this virus can also causeherpes encephalitis. Such an infection can be localized and monitoredusing a virus labeled by any of the methods described above, and variousantiviral agents can be tested for efficacy in central nervous system(CNS) infections.

[0165] Particles. Particles, including beads, liposomes and the like,constitute another entity useful in the practice of the presentinvention. Due to their larger size, particles may be conjugated with alarger number of light-generating fusion proteins than, for example, cansmall molecules. This results in a higher concentration of lightemission, which can be detected using shorter exposures or throughthicker layers of tissue. In addition, liposomes can be constructed tocontain an essentially pure targeting moiety, or ligand, such as anantigen or an antibody, on their surface. Further, the liposomes may beloaded with, for example, light-generating protein molecules, torelatively high concentrations.

[0166] Furthermore, two types of liposomes may be targeted to the samecell type such that light is generated only when both are present. Forexample, one liposome may carry luciferase, while the other carriesluciferin. The liposomes may carry targeting moieties, and the targetingmoieties on the two liposomes may be the same or different. Viralproteins on infected cells can be used to identify infected tissues ororgans. Cells of the immune system can be localized using a single ormultiple cell surface markers.

[0167] The liposomes are preferably surface-coated, e.g., byincorporation of phospholipid—polyethyleneglycol conjugates, to extendblood circulation time and allow for greater targeting via thebloodstream. Liposomes of this type are well known.

[0168] Cells. Cells, both prokaryotic and eukaryotic, constitute anotherentity useful in the practice of the present invention. Like particles,cells can be loaded with relatively high concentrations oflight-generating moieties, but have the advantage that thelight-generating moieties can be provided by, for example, aheterologous genetic construct used to transfect the cells. In addition,cells can be selected that express “targeting moieties”, or moleculeseffective to target them to desired locations within the subject.Alternatively, the cells can be transfected with a vector constructexpressing an appropriate targeting moiety.

[0169] The cell type used depends on the application. For example,bacterial cells can be used to study the infective process, and toevaluate the effects of drugs or therapeutic agents on the infectiveprocess with a high level of temporal and spatial resolution. Bacterialcells constitute effective entities. For example, they can be easilytransfected to express a high levels of a light-generating fusionprotein, as well as high levels of a targeting protein. In addition, itis possible to obtain E. coli libraries containing bacteria expressingsurface-bound antibodies which can be screened to identify a colonyexpressing an antibody against a selected antigen (Stratagene, La Jolla,Calif.). Bacteria from this colony can then be transformed with a secondplasmid containing a gene for a light-generating protein, andtransformants can be utilized in the methods of the present invention,as described above, to localize the antigen in a mammalian host.

[0170] Pathogenic bacteria can be conjugated to a light-generatingfusion protein and used in an animal model to follow the infectionprocess in vivo and to evaluate potential anti-infective drugs, such asnew antibiotics, for their efficacy in inhibiting the infection.

[0171] Eukaryotic cells are also useful as entities in aspects of thepresent invention. Appropriate expression vectors, containing desiredregulatory elements, are commercially available. The vectors can be usedto generate constructs capable of expressing desired light-generatingproteins in a variety of eukaryotic cells, including primary culturecells, somatic cells, lymphatic cells, etc. The cells can be used intransient expression studies, or, in the case of cell lines, can beselected for stable transformants.

[0172] Expression of the light-generating protein in transformed cellscan be regulated using any of a variety of selected promoters. Forexample, if the cells are to be used as light-emitting entities targetedto a site in the subject by an expressed ligand or receptor, aconstitutively-active promoter, such as the CMV or SV40 promoter may beused. Cells transformed with such a construct can also be used to assayfor compounds that inhibit light generation, for example, by killing thecells.

[0173] Alternatively, the transformed cells may be administered suchthey become uniformly distributed in the subject, and express thelight-generating fusion protein only under certain conditions, such asupon infection by a virus or stimulation by a cytokine. Promoters thatrespond to factors associated with these and other stimuli are known inthe art. In a related aspect, inducible promoters, such as the Tetsystem can be used to transiently activate expression of thelight-generating protein.

[0174] For example, CD4+ lymphatic cells can be transformed with aconstruct containing tat-responsive HIV LTR elements, and used as anassay for infection by HIV. Cells transformed with such a construct canbe introduced into SCID-hu mice and used as model for human HIVinfection and AIDS.

[0175] Tumor cell lines transformed to express the light-generatingfusion protein, for example, with a constitutively-active promoter, maybe used to monitor the growth and metastasis of tumors. Transformedtumor cells may be injected into an animal model, allowed to form atumor mass, and the size and metastasis of the tumor mass monitoredduring treatment with putative growth or metastasis inhibitors. Tumorcells may also be generated from cells transformed with constructscontaining regulatable promoters, whose activity is sensitive to variousinfective agents, or to therapeutic compounds.

[0176] Cell Transformation. Transformation methods for both prokaryoticcells and eukaryotic cells are well known in the art. Vectors containingthe appropriate regulatory elements and multiple cloning sites arewidely commercially available (e.g., Stratagene, La Jolla, Calif., orClontech, Palo Alto, Calif.).

[0177] In another aspect, the present invention includes transgenicanimals containing a heterologous gene construct encoding alight-generating fusion protein or complex of proteins. The construct isdriven by a selected promoter, and can include, for example, variousaccessory proteins required for the functional expression of thelight-generating protein, as well as selection markers and enhancerelements.

[0178] Activation of the promoter results in increased expression of thegenes encoding the light-generating fusion proteins and accessoryproteins. Activation of the promoter is achieved by the interaction of aselected biocompatible entity, or parts of the entity, with the promoterelements. If the activation occurs only in a part of the animal, onlycells in that part will express the light-generating protein.

[0179] Light-generating fusion proteins are typically administered to asubject by any of a variety of methods, allowed to localize within thesubject, and imaged. Since the imaging, or measuring photon emissionfrom the subject, may last up to tens of minutes, the subject isdesirably immobilized during the imaging process. Imaging of thelight-generating polypeptide moiety involves the use of, e.g., aphotodetector capable of detecting extremely low levels oflight—typically single photon events—and integrating photon emissionuntil an image can be constructed. Examples of such sensitivephotodetectors include devices that intensify the single photon eventsbefore the events are detected by a camera, and cameras (cooled, forexample, with liquid nitrogen) that are capable of detecting singlephotons over the background noise inherent in a detection system.

[0180] Once a photon emission image is generated, it is typicallysuperimposed on a “normal” reflected light image of the subject toprovide a frame of reference for the source of the emitted photons(i.e., localize the light-generating fusion proteins with respect to thesubject). Such a “composite” image is then analyzed to determine thelocation and/or amount of a target in the subject.

[0181] Light-generating fusion proteins that have localized to theirintended sites in a subject may be imaged in a number of ways.Guidelines for such imaging, as well as specific examples, are describedbelow.

[0182] Localization of Light-generating fusion proteins. In the case of“targeted” conjugates, that is, conjugates which contain a targetingmoiety—a molecule or feature designed to localize the conjugate within asubject or animal at a particular site or sites, localization refers toa state when an equilibrium between bound, “localized”, and unbound,“free” entities within a subject has been essentially achieved. The rateat which such an equilibrium is achieved depends upon the route ofadministration. For example, a conjugate administered by intravenousinjection to localize thrombi may achieve localization, or accumulationat the thrombi, within minutes of injection. On the other hand, aconjugate administered orally to localize an infection in the intestinemay take hours to achieve localization.

[0183] Alternatively, localization may simply refer to the location ofthe entity within the subject or animal at selected time periods afterthe entity is administered. In a related aspect, localization of, forexample, injected tumors cells expressing a light-generating moiety, mayconsist of the cells colonizing a site within the animal and forming atumor mass.

[0184] By way of another example, localization is achieved when anentity becomes distributed following administration. For example, in thecase of a conjugate administered to measure the oxygen concentration invarious organs throughout the subject or animal, the conjugate becomes“localized”, or informative, when it has achieved an essentiallysteady-state of distribution in the subject or animal.

[0185] In all of the above cases, a reasonable estimate of the time toachieve localization may be made by one skilled in the art. Furthermore,the state of localization as a function of time may be followed byimaging the light-emitting conjugate according to the methods of theinvention.

[0186] The “photodetector device” used should have a high enoughsensitivity to enable the imaging of faint light from within a mammal ina reasonable amount of time, and to use the signal from such a device toconstruct an image.

[0187] In cases where it is possible to use light-generating moietieswhich are extremely bright, and/or to detect light-generating fusionproteins localized near the surface of the subject or animal beingimaged, a pair of “night-vision” goggles or a standard high-sensitivityvideo camera, such as a Silicon Intensified Tube (SIT) camera (e.g.Hammamatsu Photonic Systems, Bridgewater, N.J.), may be used. Moretypically, however, a more sensitive method of light detection isrequired.

[0188] In extremely low light levels the photon flux per unit areabecomes so low that the scene being imaged no longer appears continuous.Instead, it is represented by individual photons which are bothtemporally and spatially distinct form one another. Viewed on a monitor,such an image appears as scintillating points of light, eachrepresenting a single detected photon. By accumulating these detectedphotons in a digital image processor over time, an image can be acquiredand constructed. In contrast to conventional cameras where the signal ateach image point is assigned an intensity value, in photon countingimaging the amplitude of the signal carries no significance. Theobjective is to simply detect the presence of a signal (photon) and tocount the occurrence of the signal with respect to its position overtime.

[0189] At least two types of photodetector devices, described below, candetect individual photons and generate a signal which can be analyzed byan image processor. Reduced-Noise Photodetection Devices achievesensitivity by reducing the background noise in the photon detector, asopposed to amplifying the photon signal. Noise is reduced primarily bycooling the detector array. The devices include charge coupled device(CCD) cameras referred to as “backthinned”, cooled CCD cameras. In themore sensitive instruments, the cooling is achieved using, for example,liquid nitrogen, which brings the temperature of the CCD array toapproximately −120° C. “Backthinned” refers to an ultra-thin backplatethat reduces the path length that a photon follows to be detected,thereby increasing the quantum efficiency. A particularly sensitivebackthinned cryogenic CCD camera is the “TECH 512”, a series 200 cameraavailable from Photometrics, Ltd. (Tucson, Ariz.).

[0190] “Photon amplification devices” amplify photons before they hitthe detection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

[0191] Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification. An exemplary microchannel intensifier-based single-photondetection device is the C2400 series, available from Hamamatsu.

[0192] Image Processors process signals generated by photodetectordevices which count photons in order to construct an image which can be,for example, displayed on a monitor or printed on a video printer. Suchimage processors are typically sold as part of systems which include thesensitive photon-counting cameras described above, and accordingly, areavailable from the same sources. The image processors are usuallyconnected to a personal computer, such as an IBM-compatible PC or anApple Macintosh (Apple Computer, Cupertino, Calif.), which may or maynot be included as part of a purchased imaging system. Once the imagesare in the form of digital files, they can be manipulated by a varietyof image processing programs (such as “ADOBE PHOTOSHOP”, Adobe Systems,Adobe Systems, Mt. View, Calif.) and printed.

[0193] The Detection Field Of The Device is defined as the area fromwhich consistent measurements of photon emission can be obtained. In thecase of a camera using an optical lens, the detection field is simplythe field of view accorded to the camera by the lens. Similarly, if thephotodetector device is a pair of “night vision” goggles, the detectionfield is the field of view of the goggles.

[0194] Alternatively, the detection field may be a surface defined bythe ends of fiber-optic cables arranged in a tightly-packed array. Thearray is constructed to maximize the area covered by the ends of thecables, as opposed to void space between cables, and placed in closeproximity to the subject. For instance, a clear material such asplexiglass can be placed adjacent the subject, and the array fastenedadjacent the clear material, opposite from the subject.

[0195] The fiber-optic cable ends opposite the array can be connecteddirectly to the detection or intensifying device, such as the input endof a microchannel intensifier, eliminating the need for a lens. Anadvantage of this method is that scattering and/or loss of photons isreduced by eliminating a large part of the air space between the subjectand the detector, and/or by eliminating the lens. Even ahigh-transmission lens transmits only a fraction of the light reachingthe front lens element.

[0196] With higher-intensity LGPs, photodiode arrays may be used tomeasure photon emission. A photodiode array can be incorporated into arelatively flexible sheet, enabling the practitioner to partially “wrap”the array around the subject. This approach also minimizes photon loss,and in addition, provides a means of obtaining three-dimensional imagesof the bioluminescence. Other approaches may be used to generatethree-dimensional images, including multiple detectors placed around thesubject or a scanning detector or detectors.

[0197] It will be understood that the entire animal or subject need notnecessarily be in the detection field of the photodetection device. Forexample, if one is measuring a light-emitting conjugate known to belocalized in a particular region of the subject, only light from thatregion, and a sufficient surrounding “dark” zone, need be measured toobtain the desired information.

[0198] Immobilizing the subject. In those cases where it is desired togenerate a two-dimensional or three-dimensional image of the subject,the subject may be immobilized in the detection field of thephotodetection devices during the period that photon emission is beingmeasured. If the signal is sufficiently bright that an image can beconstructed from photon emission measured in less than about 20milliseconds, and the subject is not particularly agitated, no specialimmobilization precautions may be required, except to insure that thesubject is in the field of the detection device at the start of themeasuring period.

[0199] If, on the other hand, the photon emission measurement takeslonger than about 20 msec, and the subject is agitated, precautions toinsure immobilization of the subject during photon emission measurement,commensurate with the degree of agitation of the subject, need to beconsidered to preserve the spatial information in the constructed image.For example, in a case where the subject is a person and photon emissionmeasurement time is on the order of a few seconds, the subject maysimply be asked to remain as still as possible during photon emissionmeasurement (imaging). On the other hand, if the subject is an animal,such as a mouse, the subject can be immobilized using, for example, ananesthetic or a mechanical restraining device.

[0200] In cases where it is desired to measure only the total amount oflight emanating from a subject or animal, the subject does notnecessarily need to be immobilized, even for long periods of photonemission measurements. All that is required is that the subject beconfined to the detection field of the photodetector during imaging. Itwill be appreciated, however, that immobilizing the subject during suchmeasuring may improve the consistency of results obtained, because thethickness of tissue through which detected photons pass will be moreuniform from animal to animal.

[0201] Further Considerations During Imaging

[0202] The visualization of fluorescent light-generating moietiesrequires an excitation light source, as well as a photodetector.Furthermore, it will be understood that the excitation light source isturned on during the measuring of photon emission from thelight-generating moiety.

[0203] Appropriate selection of a fluorophore, placement of the lightsource and selection and placement of filters, all of which facilitatethe construction of an informative image, are discussed above, in thesection on fluorescent light-generating moieties.

[0204] High-Resolution Imaging. Photon scattering by tissue limits theresolution that can be obtained by imaging LGMs through a measurement oftotal photon emission. It will be understood that the present inventionalso includes embodiments in which the light-generation of LGMs issynchronized to an external source which can be focused at selectedpoints within the subject, but which does not scatter significantly intissue, allowing the construction of higher-resolution images. Forexample, a focused ultrasound signal can be used to scan, in threedimensions, the subject being imaged. Light-generation from areas whichare in the focal point of the ultrasound can be resolved from otherphoton emission by a characteristic oscillation imparted to the light bythe ultrasound.

[0205] Constructing an Image of Photon Emission. In cases where, due toan exceptionally bright light-generating moiety and/or localization oflight-generating fusion proteins near the surface of the subject, a pairof “night-vision” goggles or a high sensitivity video camera was used toobtain an image, the image is simply viewed or displayed on a videomonitor. If desired, the signal from a video camera can be divertedthrough an image processor, which can store individual video frames inmemory for analysis or printing, and/or can digitize the images foranalysis and printing on a computer.

[0206] Alternatively, if a photon counting approach is used, themeasurement of photon emission generates an array of numbers,representing the number of photons detected at each pixel location, inthe image processor. These numbers are used to generate an image,typically by normalizing the photon counts (either to a fixed,pre-selected value, or to the maximum number detected in any pixel) andconverting the normalized number to a brightness (greyscale) or to acolor (pseudocolor) that is displayed on a monitor. In a pseudocolorrepresentation, typical color assignments are as follows. Pixels withzero photon counts are assigned black, low counts blue, and increasingcounts colors of increasing wavelength, on up to red for the highestphoton count values. The location of colors on the monitor representsthe distribution of photon emission, and, accordingly, the location oflight-generating fusion proteins.

[0207] In order to provide a frame of reference for the conjugates, agreyscale image of the (still immobilized) subject from which photonemission was measured is typically constructed. Such an image may beconstructed, for example, by opening a door to the imaging chamber, orbox, in dim room light, and measuring reflected photons (typically for afraction of the time it takes to measure photon emission). The greyscaleimage may be constructed either before measuring photon emission, orafter. The image of photon emission is typically superimposed on thegreyscale image to produce a composite image of photon emission inrelation to the subject.

[0208] If it is desired to follow the localization and/or the signalfrom a light-emitting conjugate over time, for example, to record theeffects of a treatment on the distribution and/or localization of aselected biocompatible moiety, the measurement of photon emission, orimaging can be repeated at selected time intervals to construct a seriesof images. The intervals can be as short as minutes, or as long as daysor weeks.

[0209] Analysis of Photon Emission Images Images generated by methodsand/or using compositions of the present invention may be analyzed by avariety of methods. They range from a simple visual examination, mentalevaluation and/or printing of a hardcopy, to sophisticated digital imageanalysis. Interpretation of the information obtained from an analysisdepends on the phenomenon under observation and the entity being used.

[0210] Applications: Localization of Tumor Cells

[0211] The growth and metastatic spread of tumors in a subject may bemonitored using methods and compositions of the present invention. Inparticular, in cases where an individual is diagnosed with a primarytumor, LGPs directed against the cells of the tumor can be used to bothdefine the boundaries of the tumor, and to determine whether cells fromthe primary tumor mass have migrated and colonized distal sites. Forexample, LGPs, such as liposomes containing antibodies directed againsttumor antigens and loaded with LGPs, can be administered to a subject,allowed to bind to tumor cells in the subject, imaged, and the areas ofphoton emission can be correlated with areas of tumor cells.

[0212] In a related aspect, images utilizing tumor-localizing LGPs, suchas those described above, may be generated at selected time intervals tomonitor tumor growth, progression and metastasis in a subject over time.Such monitoring may be useful to record results of anti-tumor therapy,or as part of a screen of putative therapeutic compounds useful ininhibiting tumor growth or metastasis.

[0213] In the practice of the invention, the tissue and thelight-generating fusion protein can be contacted in vitro, such as whereone or more biological samples (e.g., blood, serum, cells, tissue) arearrayed on a substrate under tissue culture conditions known by those inthe art to preserve the viability of the tissue and then the fusionprotein is added to the tissue culture. In a preferred embodiment of themethods of the invention the tissue is mammalian tissue, in particularhuman tissue.

[0214] The methods of the invention can also be practiced in vivowherein the biological sample and the light-generating fusion proteinare contacted by administration of the fusion protein (or a vectorencoding the same) to a subject suspected of containing ischemic tissueunder conditions to allow detection of the fusion protein in ischemictissue present in the subject. The invention thus also pertains to thefield of predictive medicine in which diagnostic assays, prognosticassays, pharmacogenomics, and monitoring clinical trials are used forprognostic (predictive) purposes to thereby treat an individualprophylactically. Accordingly, one aspect of the invention relates todiagnostic assays for determining the presence of ischemic tissue in abiological sample to thereby determine whether an individual isafflicted with a disease or disorder, or is at risk of developing adisorder, associated with hypoxic conditions. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is at risk of developing conditions arising from ischemia.

[0215] Referring now to the Drawings, FIG. 1A is a schematicrepresentation of different fusion proteins of the present invention.“T” indicates the enzyme target site on the fusion protein, “E”indicates the enzyme, “X” indicates the modification site on the fusionprotein, and “R” indicates the reporter domain of the fusion protein.The modification at “X” by “E” results in the fusion protein becomingactive or inactive “On/Off”. FIG. 1B shows an embodiment wherein the “T”and the “X” are separate domains and are in cis on the fusion protein.FIG. 1C shows an embodiment of the invention wherein the “T” and the “X”of the fusion protein are congruent within a domain of the fusionprotein. FIG. 1D shows an embodiment of the invention wherein the “T” ison a protein associated with the fusion protein containing the “X”.

[0216]FIG. 2 is a schematic representation of different fusion proteinsof the present invention. FIG. 2A is a schematic representation of afusion protein and associated protein of the present invention in whicha fusion protein and an associated protein “A” contains a known homo- orhetero-dimerization domain “HD” corresponding to the enzyme target siteof an enzyme, where binding of the enzyme-associated “A” to the HD onthe fusion protein results in modification of the fusion protein at “X”.FIG. 2B is a related aspect of the invention in which a binding domainof a protein “A” containing an enzyme target site “T” interacts with abinding domain “B” of a fusion protein, which results in enzyme “E”modifying the reporter domain “R” of fusion protein “B” at modificationsite “X” such that the fusion protein does or does not emit light orcause light to be emitted.

[0217]FIG. 3 shows pVHL binding to a modified form of HIF. (A) showspVHL-defective renal carcinoma cells treated with increasing amounts ofdesferoxamine (2, 10, 100, 1000 μm) or cobalt choloride (2, 10, 100,1000 μm) and immunoprecipitated with control (lane I) or anti-HIF2αantibody. Bound proteins were detected by anti-HIF2α immunoblot (IB) orby farwestern (FW) analysis with purified pVHL/elongin B/elongin C (VBC)complexes. (B) shows VBC farwestern and anti-HIF2α immunoblot analysisof ts20 cells grown at the restrictive temperature under hypoxic ornormoxic conditions. (C and D) depict GST-HIF1α (530-652), containingthe oxygen-dependent degradation domain (ODD), produced in E. Coli,recovered on glutathione Sepharose, and incubated with rabbitreticulocyte lysate for 90 min at 30° C. In (D, lane 3), thereticulocyte lysate was first heat inactivated for 20 min. Followingstringent washes the GST-ODD protein was subjected to VBC farwestern andanti-GST immunoblot analysis.

[0218]FIG. 4 shows pVHL binding to a HIF1α-derived peptide if Leu562 andPro564 are intact. (A) shows binding of the indicated ³⁵S-labeledGal4-HIF1α fusion proteins to immobilized GST-pVHL, elongin B, elongin Ccomplexes. (B) shows binding of ³⁵S-labeled pVHL to biotinylated HIF1α(556-575) peptides with the indicated substitutions of residues 561-568.‘+’ indicates preincubation of peptide with unprogrammed reticulocytelysate prior to addition of pVHL. (C and D) depict ³⁵S-labeled wild-type(WT), Pro564Ala, and Leu562Ala full-length HA-HIF1α (panel C) andGal4-HA-HIF1α (530-652)(panel D) proteins immunoprecipitated withanti-HA antibody or captured with immobilized GST-VBC complexes.WG=wheat germ extract; Retic=rabbit reticulocyte lysate.

[0219]FIG. 5 shows ubiquitination and degradation of HIF linked toLeu562 and Pro 564. (A) depicts in vitro ubiquitination of ³⁵S-labeledwild-type, Leu562A, and Pro564A Gal4-HA-HIF1α (530-652) in the presenceof S100 extracts prepared from pVHL-defective renal carcinoma cellsstably transfected to produce wild-type pVHL or with empty vector. (B)shows in vitro degradation of ³⁵S-labeled wild-type, Leu562Ala, andPro564Ala in xenopus egg extracts. (C) is an anti-HA immunoblot analysisof COS7 cells transiently transfected with 1.5 or 3.5 μg of plasmidsencoding wild-type or P564A HA-HIF1α in the absence or presence ofdesferoxamine.

[0220]FIG. 6 depicts proline hydroxylation linked to pVHL-binding. (A)is a MALDI-TOF analysis of wild-type, Pro564Ala, and Leu562Alabiotinylated HIF(556-575) peptides following incubation with rabbitreticulocyte lysate. (B) shows Gal4-HA-HIF (555-575) translated in vitroin the presence of 3H-Proline with rabbit reticulocyte lysate or wheatgerm extract and gel-band purified. Following acid hydrolysis prolineand hydroxyproline were separated using thin layer chromatography. Thedashed circle indicates positions of ninhydrin stained proline andhydroxyproline markers.

[0221]FIG. 7 illustrates that pVHL specifically recognizes HIF1α withhydroxylated proline 564. (A and B) shows binding of ³⁵S-labeled pVHL tobiotinylated HIF1α (556-575) peptides with the indicated substitutionsof residues 561-568. (C) shows ts20 cells stably transfected to produceHA-pVHL grown at restrictive (lane 1) or permissive (lanes 2-6)temperature and immunoprecipitated anti-HIF1α (lane 1 and 2) or anti-HAantibody (lanes 3-7). Bound proteins were eluted by boiling in samplebuffer (lane 1 and 2) or treatment with the indicated peptides and thenimmunoblotted with anti-HIF1α antibody. (D) shows pVHL-defective renalcarcinoma cells stably transfected to produce wild-type pVHL (WT8) orwith empty vector (RC3) metabolically labeled with ³⁵S methionine,lysed, and incubated with immobilized biotinylated HIF1α (556-575)peptides with the indicated substitutions of residue 564. Specificallybound proteins were detected by autoradiography.

[0222]FIG. 8 illustrates the production of TETr-cyclins A and E. (A) isa schematic of TETr-cyclin fusion protein. (B) shows production ofTETr-cyclin A and TETr-cyclin E. Cells transfected to produce theindicated cyclin A or cyclin E proteins were lysed and immunoblotted(IB) with the indicated antibodies. (C) shows phosphorylation of pRB byTETr-cyclins A and E in SAOS-2 cells. pRB-defective SAOS-2 cellstransfected so as to produce HA-tagged pRB along with the indicatedcyclins were lysed and immunoblotted with anti-HA antibody. The % oftransfected cells in G1 and S phase was determined by fluororescenceactivated cell sorting (FACS).

[0223]FIG. 9 shows DNA bound cyclins A and E differentially affectingtranscription. (A) is a schematic of reporter plasmid (pUHC 13-3) thatcontains seven TETracycline operator sequences (TETo) upstream of aminimal CMV promoter that includes a TATA box. (B, C) show U2OS cellswere cotransfected with plasmids encoding the indicated TETr fusionproteins along with the pUHC 13-3 reporter plasmid and a plasmidencoding β-galactosidase. Numbers shown at the bottom of the graphindicate the amount of TETr plasmid (in μg). 48 hours later luciferaseactivity, normalized for β-galactosidase, was determined. Foldrepression is the corrected luciferase value for TETr alone divided bythe corrected luciferase values for the indicated TETr fusion proteins.Fold activation represents the corrected luciferase values for theindicated TETr fusion proteins divided by the corrected luciferase valuefor TETr alone.

[0224]FIG. 10 illustrates transcriptional regulation by cyclins A and Edependent upon DNA binding. (A) shows U2OS cells cotransfected withplasmids encoding the indicated TETr fusion proteins along with the pUHC13-3 reporter plasmid and a plasmid encoding β-galactosidase. 24 hourslater doxycycline was added to a final concentration of 2 μg/ml whereindicated by a ‘+’. 24 hours later, luciferase activity, corrected forβ-galactosidase activity, was determined and expressed as foldrepression or activation relative to cells producing TETr alone. (B)shows U2OS cells cotransfected with plasmids encoding the indicatedcyclins along with the pUHC 13-3 reporter plasmid and a plasmid encodingβ-galactosidase. Fold repression and activation was determined as in(A). (C) shows U2OS cells cotransfected with plasmids encoding TETr orTETr-cyclin E, along with a minimal HSV-TK promoter reporter plasmidcontaining the indicated number of TETo binding sites and a plasmidencoding α-galactosidase. Doxycycline was added as in (A).

[0225]FIG. 11 illustrates that cyclin box is required fortranscriptional repression by DNA bound cyclin A. (A) U2OS cells werecotransfected with plasmids encoding the indicated TETr-cyclin Avariants along with the pUHC 13-3 reporter plasmid and a plasmidencoding β-galactosidase. Cell extracts were prepared and luciferaseactivity, corrected for β-galactosidase activity, was expressed as foldrepression relative to cells producing TETr alone. (B) The indicatedTETr-cyclin A variants were translated in vitro in the presence of³⁵S-methionine and incubated with GST-cdk2 and glutathione Sepharose.Specifically bound proteins were resolved by SDS-polyacrylamide gelelectrophoresis and detected by autoradiography. In parallel, 20% of theinput proteins were resolved by SDS-polyacrylamide gel electrophoresisand detected by autoradiography. (c) pRB defective SAOS-2 cellstransfected so as to produce HA-tagged pRB along with the indicatedTETr-cyclin A variants were lysed and immunoblotted with anti-HAantibody.

[0226]FIG. 12 shows transcriptional activation by cyclin E linked to itsability to bind to cdk2 and interact with substrates. (A) U2OS cellswere cotransfected with plasmids encoding the indicated TETr-cyclin Evariants along with the pUHC 13-3 reporter plasmid and a plasmidencoding β-galactosidase. Cell extracts were prepared, and luciferaseactivity, corrected for β-galactosidase activity, was expressed as foldactivation relative to cells producing TETr alone. (B) The indicatedTETr-cyclin E variants were translated in vitro in the presence of³⁵S-methionine and incubated with GST-cdk2 and glutathione Sepharose.Specifically bound proteins were resolved by SDS-polyacrylamide gelelectrophoresis and detected by autoradiography. In parallel, 20% of theinput proteins were resolved by SDS-polyacrylamide gel electrophoresisand detected by autoradiography. (c) pRB defective SAOS-2 cellstransfected so as to produce HA-tagged pRB along with the indicatedTETr-cyclin E variants were lysed and immunoblotted with anti-HAantibody.

[0227]FIG. 13 shows transcriptional activation by DNA bound cyclin Edependent on cdk2 catalytic activity. (A, B) U2OS cells were transientlycotransfected with plasmids encoding TETr-cyclin A or E and, whereindicated, increasing amounts of a plasmid encoding a dominant-negative(dn) form of cdk2. Cell extracts were prepared and luciferase activity,corrected for β-galactosidase activity, was determined. Correctedluciferase values were expressed as fold repression (A) or activation(B) relative to TETr alone. (C) U2OS cells were transiently transfectedwith plasmids encoding TETr-cdk2 or TETr-cdk2 (N132A) and a plasmidencoding either cyclin A or E. Cell extracts were prepared andluciferase activity, corrected for β-galactosidase activity, wasdetermined. Corrected luciferase values were expressed as foldactivation relative to TETr alone.

[0228]FIG. 14 shows transcriptional effects mediated by cell-cycledependent changes in endogenous cyclins E and A. (A,B) 3T3 cells stablytransfected with a luciferase reporter plasmid containing 7 TETo sites(pUHC 13-3) and a plasmid encoding TETr-cdk2 were serum-starved for 72hours and subsequently re-fed with serum. At various timepointsthereafter aliquots of cells were removed and either lysed forimmunoblot analysis with the indicated antibodies or analyzed for DNAcontent by propidium iodide staining followed by fluorescence activatedcell sorting (FACS). (C) 3T3 cells stably transfected with a luciferasereporter plasmid containing 7 TETo sites (pUHC 13-3) in the absence(open circles) or presence of a plasmid encoding TETr-cdk2 (closedsquares) or TETr-cdk2 (N132A) (open squares) were serum-starved for 72hours and then re-fed with serum in the presence or absence ofdoxycycline. At various timepoints thereafter luciferase assays wereperformed. To correct for general effects due to serum, the luciferasevalues at each timepoint in the absence of doxycycline were corrected bysubtracting the luciferase assay obtained in the presence ofdoxycycline. After correction, the luciferase values for the two cellpopulations were expressed relative to the corresponding luciferasevalues obtained at time 0. In parallel, the cells producing TETr-cdk2were lysed and immunoprecipitated with anti-cyclin A or anti-cyclin Eantibodies. The immunoprecipitates were then used to phosphorylateHistone H 1 in vitro.

[0229] Methods of Treating Hypoxia or Ischemia Related Tissue Damage andModulating Angiogenesis or Vascularization. Tissue ischemia is a majorcause of morbidity and mortality. In principle, drugs that stabilize HIFmay augment angiogenesis and the adaptation of hypoxia. The activationof HIF by hypoxia is complex and involves protein stability, nuclearlocalization, DNA binding capability and transcriptional activationfunction. The discovery that proline hydroxlaytion governs HIF turnoverin the presence of oxygen will facilitate the dissection of themechanism underlying the various aspects of HIF regulation.

[0230] The invention also provides various methods of treating, i.e.,reducing, preventing or delaying the onset of HIF-1 related disorders,modulating angiogenesis or vascularization. Examples of HIF-1 mediateddisorders include chronic and acute hypoxia or ischemia relateddisorders such as tissue damage and scarring. Acute hypoxia or ischemiarelated disorders include for example myocardial infarction, stroke,cancer and diabetes such as tissue damage and scarring. Chronic hypoxiaor ischemia related disorders include for example, deep vein thrombosis,pulmonary embolus and renal failure.

[0231] In one aspect the invention the invention provides methods oftreating or preventing a hypoxic or ischemic related disorder ormodulating angiogenesis or vascularization by administering to a subjecta compound that decreases prolyl hydroxylase, expression or activity.Examples of prolyl hydroxylase includes human Egl-9 or homologs.(Epstein, et al Cell 107:43-54, 2001) The compound can be a prolylhydroxylase antibody, a nucleic acid that decreases the expression of anucleic acid that encodes a prolyl hydroxylase polypeptides such as aprolyl hydroxylase anti-sense nucleic acid or a compound identified byany of the methods of the invention. Preferably, the half life of HIF inthe subject is increased in the presence of the compound as compared tothe absence of the compound.

[0232] In a further aspect the invention includes a method of treatingcancer in a subject by administering to the subject a compound thatincreases prolyl hydroxylase expression or activity. Preferably, thecompound is a compound that has been identified by the methods of theinvention.

[0233] In another aspect the invention provides a method for treating orpreventing a hypoxia or ischemic related disorder in a subject byadministering to a subject a compound that which modulates prolylhydroxylation of HIF.

[0234] In still a further aspect the invention provides a method fortreating or preventing a HIF related disorder by administering to asubject a compound that which modulates prolyl hydroxylation of HIF suchthat the HIF related disorder is prevented reversed of stabilized.

[0235] In yet another aspect the invention provides a method forregulating HIF turnover in a subject by administering to a subject acompound that which modulates prolyl hydroxylation.

[0236] By “modulates” is meant to increase or decrease the prolylhydroxylation of HIF. Compounds that inhibits prolyl hydroxylationinclude several small molecule proline hydroxylase inhibitors which havebeen developed as antifibrotic agents.

[0237] The subject is preferably a mammal. The mammal can be, e.g., ahuman, non-human primate, mouse, rat, dog, cat, horse, or cow. Invarious aspects the subject include patients with coronary, cerebral, orperipheral arterial disease and patients with one or more non-healingwounds.

EXAMPLES Example 1 Characterization of Hypoxia-Responsive Polypeptides

[0238] In order to demonstrate the efficacy of the First embodiment ofthe invention, e.g. employing a hypoxia-responsive LGP, the interactionof pVHL and HIF was examined. A HIF1α polypeptide that is sufficient tobind pVHL is disclosed herein. pVHL binds directly to a region of HIF1αcalled the oxygen-dependent degradation domain (ODD). pVHL recognizesHIF produced in rabbit reticulocyte lysate but not HIF produced in wheatgerm extracts or in E. Coli. Furthermore, wheat germ or E. Coli-derivedHIF binds to pVHL following preincubation with a human, rabbit, orxenopus cell extracts at 37° C. For example, glutathioneS-transferase-ODD fusion proteins produced in E. Coli were notrecognized by VBC in farwestern assays (FIG. 3C). These proteins wererecognized, however, after pre-incubation with a rabbit reticulocytelysate. Similar results were obtained with GST-ODD fusion proteins ofvarious sizes, thus excluding the possibility that the farwestern blotsignal represents a spurious interaction between VBC and areticulocyte-derived protein. VBC did not recognize GST-ODD fusionproteins incubated with a heat-inactivated reticulocyte lysate (FIG.3D). Gal4-HIF fusion proteins containing HIF residues 555-575 boundspecifically to immobilized GST-VHL, elongin B, elongin C complexes(FIG. 4A). Coupled in vitro transcription/translation of ³⁵S-labeledproteins was conducted according to the manufacturer's instructions(TNT, Promega). Also, a biotinylated peptide corresponding to HIFresidues 556-575 bound to pVHL following pre-incubation withreticulocyte lysate (FIG. 4B). For peptide binding studies, 1 μg ofbiotinylated peptide was bound to 30 μl of monomeric avidin Sepharose(Pierce). Where indicated, the peptide was pre-incubated with 50 μl ofrabbit reticulocyte lysate for 90 min at 30° C. The Sepharose was thenwashed 3 times with NETN (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA,0.5% non-idet P40) and used in binding reactions containing 4 μl³⁵S-HA-pVHL in 500 μl of EBC or 500 μl ³⁵S-radiolabeled cell extract(equivalent to cells from a subconfluent 100 mm dish). Following 1 hourincubation at 4° C. with rocking the Sepharose was washed 4 times withNETN. Bound proteins were eluted by boiling in SDS-containing samplebuffer and detected by autoradiography. This region of HIF contains ahighly conserved 8 mer (MLAPYIPM) which, when mutated to 8 consecutivealanines, leads to HIF stabilization in cells. An alanine scan of thisregion showed that Leu562 and Pro564 were essential for specific bindingto pVHL in this assay (FIG. 4B). In contrast, mutation of the onepotential phosphoacceptor in this peptide, Tyr565, did not affect pVHLbinding, in keeping with an earlier study in which a Tyr565Phe mutationdid not affect HIF stability. In addition, the binding of pVHL toGST-ODD in the assays described above was unaffected by phosphatasetreatment.

[0239] Binding of pVHL to HIF1α is critically dependent upon residuesLeu562 or Pro564 of HIF1α. Importantly, mutation of either Leu562 orPro564 in the context of full-length HIF1α or a Gal4-ODD fusion proteinalso led to a loss of pVHL binding activity (FIGS. 4C and 4D,respectively). Gal4-ODD made with reticulocyte lysate contained anelectrophoretically distinct band compared with Gal4-ODD made with wheatgerm extract (FIG. 4D). This electrophoretically distinct protein boundto VBC and was undetectable among the Leu562Ala and Pro564Alatranslation products. The isoelectric points of the two arrowed bands inFIG. 4D were identical following 2-D gel electrophoresis indicating thatthe putative modification did not involve a change in protein charge.

[0240] Modification of HIF1α by pVHL following binding is alsocritically dependent upon residues Leu562 or Pro564 of HIF1α. Gal4-HIFfusion proteins with the Leu562Ala mutation or Pro564Ala mutationdisplayed diminished pVHL-dependent polyubiquitination in vitro relativeto the corresponding wild-type protein (FIG. 5A). Qualitatively similarresults were obtained with the corresponding full-length HIF1α species,although this assay is less robust than with the Gal4-ODD fusionproteins. Likewise, HIF1 αPro564Ala and HIF1α Leu562Ala were far morestable than wild-type HIF1α in in vitro degradation assays performedwith xenopus extracts (FIG. 5B). Xenopus egg extracts were made as iswell known in the art and stored frozen until use. Degradation reactionscontained 8 μl of egg extract, 0.1 μl of 100 mg/ml cyclohexamide, 0.25μl of energy regeneration mix, 0.25 μl of bovine ubiquitin, and 0.4 μlof ³⁵S-radiolabeled HIF and were carried out at room temperature. At theindicated timepoints 1 μl aliquots were removed and placed in samplebuffer. Samples were resolved on 5-15% gradient gels and analyzed byautoradiography. Biotinylated HIF (556-575) peptides were incubated withrabbit reticulocyte lysate, washed, eluted with free biotin, andanalyzed by mass spectrometry. 1 μg of HPLC-purified peptide was boundto 30 PI monomeric avidin Sepharose and incubated with 100 μl of rabbitreticulocyte lysate at room temperature for 1 hour with tumbling.Following brief centrifugation the reticulocyte lysate was removed,fresh reticulocyte lysate was added, and the cycle was repeated 6 times.The Sepharose was then washed 4 times with NETN and once with PBS. Themodified peptide was eluted in 50 μl of 20 mM ammonium acetate [pH 7.0],2 mM biotin. In these experiments Met561 and Met568 were converted toalanine to prevent spurious oxidation of the methionine residues duringanalysis. This double alanine substitution, like the correspondingsingle substitutions, did not affect pVHL binding. Treatment of the HIF(556-575) peptide with rabbit reticulocyte lysate led to the appearanceof a second peak in MALDI-TOF assays representing an increase inmolecular weight of 16 (FIG. 6A). This peak was not detectable prior toincubation with reticulocyte lysate and was not detected in thecorresponding reticulocyte-treated Leu562Ala and Pro564Ala peptides(FIG. 6A). Postsource decay analysis using the same instrument placedthe addition of +16 at Pro 564. Similarly, electrospray ion trap massspectrometry/mass spectrometry (MS/MS) analysis was consistent withaddition of +16 at Pro 564 and excluded such a modification of Leu 562.Finally, MS/MS analysis of the reticulocyte-treated HIF (556-575)peptide produced a pattern of ions that was identical to the patternobtained with a HIF (556-575) peptide which was synthesized to containhydroxyproline at position 564. Next, Gal4-HIF(555-575) was translatedin vitro in the presence of ³H-Proline using rabbit reticulocyte lysateor wheat germ extract, gel-band purified, and subjected to acidhydrolysis and thin layer chromatography. 2 ml of ³H—P-labeled Gal4-HIF(555-575) in vitro translate was immunoprecipitated with 50 μg ofanti-HA antibody (12CA5, Roche), resolved on a 12% SDS-polyacrylamidegel, and transferred to a PVDF membrane. Gal4-HIF (555-575) wasvisualized by autoradiography and the corresponding region of PVDF wasexcised, hydrolyzed by incubation in 100 μl of 10 N HCl at 105° C. for 3hours. Samples were evaporated to dryness, resuspended in 20 μl H₂Ocontaining 10 μg of unlabeled proline and 4-OH proline (Sigma), andresolved by 2-D thin layer chromatography using phenol-distilled H₂O inthe first dimension and N-butanol-acetic acid-H₂O in the second.Following visualization of standards with ninhydrin radiolabeled prolinewas detected by autoradiography. Gal4-HIF produced in rabbitreticulocyte lysate, but not in wheat germ, contained hydroxyproline(FIG. 6B).

[0241] pVHL specifically recognizes a proline hydroxylated determinant.The HIF (556-575) peptide containing hydroxyproline at position 564bound to pVHL with or without pretreatment with reticulocyte lysate(FIG. 7A). The mass spectrometry analysis of the Leu562Ala peptideshowed that Leu562 was required for HIF modification (FIG. 6A). Indeed,pVHL bound to a HIF (556-575) peptide with the Leu562Ala substition andhydroxyproline at residue 564 (FIG. 7B). This suggests that the primaryrole of Leu562 is to allow for the hydroxylation of Pro 564. Twoapproaches were used to demonstrate that hydroxylated HIF peptide couldinteract with cell-derived pVHL complexes. Ts20 cells were engineered toproduce HA-tagged pVHL which carry a temperature-sensitive mutation inthe E1-ubiquitin activating enzyme. ts20 cells were transfected withpIRES-HA-VHL, pIRES-HA-VHL (Y98H), or pIRES-Neo (Invitrogen) andselected in the presence of 1 mg/ml G418. Individual G418-resistantcolonies were isolated using cloning cylinders and expanded. Cellsproducing HA-VHL or HA-VHL (Y98H) were identified by anti-HA immunoblotanalysis. HIF coimmunoprecipitated with HA-pVHL at the restrictivetemperature. HIF bound to pVHL in this way could be eluted by thehydroxylated HIF (556-575) peptide but not the unmodified peptide (FIG.7C). For the tests shown in FIG. 7C, ts20 cells were grown at therestrictive or permissive temperature for 14 hours, methionine-starvedfor 90 min, and then grown in the methionine-free media supplementedwith ³⁵S-met (500 mCi/ml) for 90 min. Cells were washed once with coldPBS, lysed in EBC, and immunoprecipitated with anti-HA (12CA5; Roche oranti-HIF1α (NB100-105; Novus).

[0242] Following 5 washes with NETN bound proteins were eluted byboiling in sample buffer or by incubation in 65 μl of PBS containing 7μg of the indicated peptide. Moreover, HIF was not eluted by the HIF(556-575) Pro564Ala peptide or by a poly-hydroxyproline peptide (FIG.7C). Affinity chromatography was performed with immobilized peptides andmetabolically labeled matched renal carcinoma cells which do (WT8) or donot (RC3) produce HA-pVHL. 786-0 subclones were starved for 1 hour,grown in the methionine-free media supplemented with ³⁵S-met (500mCi/ml) for 3 hr, washed once with ice cold PBS, and lysed in EBC. Theproline hydroxylated HIF (556-575) peptide specifically bound to pVHL aswell as proteins with the expected electrophoretic mobilities of thepVHL-associated proteins elongin B, elongin C, and Cul2 (FIG. 7D). Theidentity of pVHL in this experiment was confirmed by western blotanalysis. Likewise, the binding of endogenous pVHL to the hydroxylatedpeptide was detected using 293 embryonic kidney cells. A HIF1α mutant inwhich Proline 564 was converted to alanine was stabilized in cells andinsensitive to the hypoxia-mimetic desferoxamine (FIG. 5C).

[0243] The HIF1α (protein is stabilized in hypoxic conditions andfunctions to inhibit, decrease and/or reverse hypoxia in affectedtissues, e.g. in a solid tumor, diabetic retinopathy or ischemic hearttissue, in part by modulating pro-angiogenic factors. A light-generatingfusion protein containing a HIF1α moiety will also be stabilized inhypoxic tissue. Destruction of a HIF1α protein or a HIF1α-containing LGPvia the pVHL ubiquitin pathway occurs, e.g. during normoxia, when aprolyl hydroxylase modifies the HIF1α protein such that pVHL binds toand modifies HIF1α. This modification process acts as a dynamic switchto regulate the bioluminescence of the HIF1α-containing light-generatingfusion protein such as the First embodiment of the invention. AHIF1α-containing light-generating fusion protein is useful todynamically image hypoxic tissues, e.g. cancer, in situ, and to screenfor or test the efficacy of hypoxia-modulating compounds.

Example 2

[0244] The following example demonstrates the efficacy oflight-generating fusion proteins of the invention for imaging hypoxictissues.

[0245] The Oxygen-Dependent Degradation Domain (ODD) of HIF1α, rendersHIF1α unstable in the presence of oxygen. This region is recognized bypVHL. pVHL specifically binds directly to peptidic determinants,corresponding to HIF1α residues 555-575, located within the ODD. At thecore of this peptide there is a conserved proline residue (residue 564)which, in the presence of oxygen, becomes enzymatically hydroxylated.This residue serves as the signal for pVHL to bind. In sum, in thepresence of oxygen, the HIF peptide becomes hydroxylated and isrecognized by pVHL. In the absence of oxygen (hypoxia), the modificationdoes not take place and pVHL does not bind to HIF.

[0246] Construction of ODD-LUC Plasmids

[0247] In a first set of experiments the ability of a small HIF peptide(555-575) to function in cis to target a foreign protein forpVHL-dependent proteolysis was evaluated. To this end, the HIF cDNAencoding amino acids 555-575 (hereafter ‘ODD’) was PCR amplified witholigonucleotides that introduced convenient restriction sites, digested,and gel-band purified. The PCR fragment was then subcloned, in-frame, 3′of a Firefly luciferase cDNA contained in the pGL3 plasmid (Promega).The resulting luciferase-ODD chimeric cDNA was subcloned into pcDNA3(Invitrogen) to facilitate in vitro translation and expression studiesin mammalian cells. In parallel a pcDNA3 plasmid encoding wild-typeFirefly luciferase and pcDNA3 plasmid encoding wild-type HIF1 α was usedas controls.

[0248] VBC-GST Pulldown of ODD-Luciferase

[0249] To determine whether the ODD-luciferase protein could bind topVHL, GST-VHL, elongin B, and elongin C (‘GST-VBC’) were produced in E.Coli and recovered as a trimeric complex on glutathione sepharose.Earlier work showed that pVHL does not fold properly in the absence ofelongin B and elongin C. HIF1α, ODD-luciferase, and wild-type luciferasewere translated in vitro using rabbit reticulocyte lysate (RRL) or wheatgerm extract (WG) in the presence of ³⁵S methionine (FIG. 15). Aliquotsfrom the in vitro translation reactions were added to recombinantVBC-GST prebound to glutathione sepharose and incubated for 1 hour. Thesepharose was then washed and bound proteins were resolved on a 12% SDSpolyacrylamide gel. The gel was then dried and exposed to film. pVHLbound to reticulocyte-generated HIF1α and ODD-Luc, but not Luc (comparelanes 4, 8 and, 12). Furthermore, pVHL did not bind to wheat germproduced ODD-Luc, as wheat germ lacks the HIF prolyl hydroxylase(compare lanes 6 and 8). Thus the data show that ODD-Luc wasspecifically recognized by pVHL.

[0250] Peptide Competition Assay

[0251] The observation that pVHL bound to ODD-Luc produced in RRL, butnot WG, strongly suggested that the ODD-Luc, like HIF, needed to undergoprolyl hydroxylation in order to be recognized by pVHL. To study thisfurther, the GST-VBC binding experiments were repeated in the presenceof synthetic HIF (555-575) peptides in which Pro 564 was hydroxylated(P-OH) or was not hydroxylated (P). GST-VBC was mixed with either thecontrol (P) peptide or the P-OH peptide in increasing concentrations(from 0-5 μg). Next in vitro translated (in retic lysate) Luc, ODD-Luc,or HIF1α was then added to see if it could efficiently compete off anybound P-OH. HIF and ODD-Luc were able to compete off the P-OH peptideeffectively at even very high concentrations of P-OH. Thus, the resultsobtained with ODD-Luc recapitulated earlier binding studies performedwith authentic HIF. (FIG. 16.)

[0252] Luciferase Activity in Cells

[0253] In pilot experiments, it was confirmed that ODD-Luc in vitrotranslate retained luciferase activity in vitro comparable to wild-typeluciferase. To begin to ask whether the ODD-Luc was oxygen sensitive incells, transient transfection assays were performed. In the first set ofexperiments, 100 mm tissue culture plates were seeded with 8×10⁵ HeLacells per plate. Eighteen hours later the cells were transientlytransfected, using lipofectamine (Gibco), with the pcDNA3 plasmidsencoding ODD-Luc or wild-type Luc along with an internal control plasmidencoding Renilla luciferase. Twenty-four hours after transfection thecells were split into 6 well plates and allowed to adhere and grow for8-12 hours. At this point the hypoxia mimetics desferrioxamine (DFO) orcobalt chloride (CoCl2) were added directly to the media at finalconcentrations of 500 mM and 200 mM respectively, in duplicate wells. Inaddition, some wells were not treated so as to serve as controls. 12hours after treatment with DFO and CoCl2 the cells were lysed withPassive Lysis Buffer (Promega), rocked at room temperature for 20minutes, and assayed for Firefly and Renilla Luciferase according to themanufacturers instructions. Results were obtained in duplicate andaveraged. (FIG. 17)

[0254] In the untreated cells the luciferase values for ODD-Luc wereapproximately 30% of the values obtained with wild-type Luc. Note thatHeLa cells have wild-type pVHL and these experiments were conductedusing cells grown in the presence of oxygen. The addition ofhypoxia-mimetics led to a marked increased in ODD-Luc luciferaseactivity, whereas no such induction was detected with wild-typeluciferase. The induced levels of ODD-luciferase were comparable tothose obtained with wild-type luciferase. These results are consistentwith the idea that ODD-Luc is subject to pVHL-dependent proteolysis incells whereas wild-type Luc is not.

[0255] Imaging of ODD-Luc in Xenograft Tumors in Nude Mice.

[0256] To verify in a mammalian system that the ODD-Luc gene is indeedregulated by hypoxia or hypoxia-mimetics, the following experiment wasconducted. Given the ease of subcutaneously transplanting tumors andtheir ability to grow and vascularize, the ability to image luciferaseactivity of tumors at the subcutaneous level and assess ODD-Luc'sresponse to hypoxia was tested as follows.

[0257] Polyclonal Hela cells stably transfected with ODD-Luc and Lucwere generated. The clones were suspended in PBS and counted. 10⁶ cellsper injection site were administered subcutaneously in duplicate intothe flanks of nude mice (FIG. 18). Upon growth of palpable tumors(approximately 3-4 days) the mice were given intraperitoneal injectionsof phenobarbital for anesthesia, injected with a weight-adjusted dose ofluciferin, and whole body imaged with a Xenogen Camera. To ensure thatthe difference in luciferase activity was not simply a function of tumorsize alone, bidimensional tumor measurements were taken and wereapproximately equal.

[0258]FIG. 18 shows three different mice injected with ODD-Luc (right)and Luc (left) in duplicate. The sites where ODD-Luc was injectedclearly have attenuated luciferase signal. As such, the data show a realattenuation of luciferase activity, secondary ubiquitination anddestruction of ODD-Luc.

Example 3 Light-Generating Fusion Proteins Including a DNA Binding Site

[0259] In order to demonstrate the efficacy of the Second embodiment ofthe invention, a LGP capable of interacting with nucleic acids in orderto kinetically monitor gene transcription in vivo, in vitro or insilico, the impact of cyclins on transcription was examined. As it isuseful to monitor either the induction or repression of transcription,cyclins with specific affects on transcription were investigated.

[0260] Fusion of a DNA-binding motif to a cyclin does not alter cyclinbinding to a cdk or the kinase activity of the cyclin/cdk complex.Mammalian expression plasmids were generated that encode fusion proteinsconsisting of the TET repressor DNA-binding domain (TETr) (Gossen andBujard, 1992) fused to cyclin A or cyclin E with an intervening flexiblelinker consisting of Gly4-Ser repeats (FIG. 8A). Both of these plasmidsgave rise to stable proteins of the expected size following transfectioninto mammalian cells (FIG. 8B). TETr-cyclin A and TETr-cyclin E, liketheir unfused counterparts, bound to cdk2 (FIG. 1 1 and 12) and couldphosphorylate p107 in vitro. Furthermore, both TETr-cyclin A andTETr-cyclin E promoted pRB phosphorylation and bypassed a pRB-inducedGI/S block when cointroduced with wild-type pRB into pRB-defective tumorcells (FIG. 8C).

[0261] Cyclin A and cyclin E dramatically affect transcription. U2OScells were transiently transfected with plasmids encoding various TETrfusion proteins and a luciferase reporter plasmid containing 7 TETobinding sites upstream of a TATA box derived from the CMV promoter (FIG.9A). TETr binds specifically to TETo sites. As expected, TETr-RBrepressed transcription from this reporter plasmid whereas TETr-E2 μlactivated the reporter (FIG. 9B-C). The basal activity observed withthis reporter plasmid presumably reflects the presence of crypticenhancer sequences. In this and subsequent assays, the TETr domain alonewas essentially inert. Surprisingly, TETr-cyclin A and TETr-cyclin Eboth dramatically affected transcription in this assay and did so inopposite ways. TETr-cyclin A decreased transcription approximately 80%(5-fold repression) whereas TETr-cyclin E increased transcription10-fold (FIG. 9B-C). Doxycycline prevents the binding of TETr to TEToand completely blocked the transcriptional effects of TETr-cyclin A andTETr-cyclin E (FIG. 10A). As expected, doxycycline also blocked thetranscriptional effects of TETr-RB and TETr-E2F1, which were tested inparallel. Furthermore, unfused cyclin A and E had no effects on theTETo-driven reporter plasmid (FIG. 10B). Experiments were repeated usingreporters containing 1, 2, 3, or 7 TETo in which the CMV-derived TATAbox was replaced with a minimal HSV TK promoter (Gossen and Bujard,1992) (FIG. 1C). TETr-cyclin E also activated these reporters indoxycycline-inhibitable manner. The degree of activation observed withthe HSV TK series of reporters was lower than with the CMV TATA-basedreporter, in keeping with earlier results obtained with these reportersand fused TETr to the HSV VP 16 transcriptional activation domain(Gossen and Bujard). The low basal level of transcription from thesereporters precluded analysis of repression by cyclin A.

[0262] The specific domains of the cyclins that bind to the cdks arecritical for cyclin-mediated transcriptional regulation. Plasmidsencoding TETr fused to various colinear fragments of cyclin A and E wereused to determine which regions of these molecules are required fortranscriptional regulation (FIGS. 11 and 12). All of the resultingfusion proteins were expressed at comparable levels in transienttransfection experiments. Cyclin A (1-310), like wild-type cyclin A,repressed transcription when fused to TETr (FIG. 11A). This fragment ofcyclin A does not bind to cdk2 (FIG. 11B) and cannot direct thephosphorylation of pRB when introduced into cells (FIG. 11C).Conversely, a cyclin A point mutant (E220A) (Schulman et al., 1998) thatmeasurably interacts with cdk2 (FIG. 11B) and directs thephosphorylation of pRB (FIG. 11C) did not repress transcription in theseassays (FIG. 11A). This mutation maps to the cyclin A cyclin box (FIG.11A). TETr-cyclin A also repressed transcription when tested in p107−/−; p130 −/− mouse fibroblasts and cyclin A (1-310) does not bind toeither p107 or p130. Only those cyclin E mutants that bind to cdk2 (FIG.12B) and could direct the phosphorylation of pRB (FIG. 12C) scored astranscriptional activators (FIG. 12A). For example, Schulman et al(1998) identified cyclin A residues that are critical for substratebinding and assembly with cdk2. Mutation of analogous residues in cyclinE produced a mutant (cyclin E L134A/Q174A) that likewise failed to bindto cdk2 (FIG. 12B) and failed to phosphorylate pRB (FIG. 12C). Thismutant did not activate transcription (FIG. 12). In keeping with theseresults, a dominant-negative form of cdk2 blocked transcriptionalactivation by cyclin E (FIG. 13B) but had no effect on transcriptionalrepression by cyclin A (FIG. 13A). Similarly, cyclin E, but not cyclinA, activated transcription in concert with a TETr-cdk2 fusion providedthe kinase domain was intact (FIG. 13C). Comparable production ofTETr-cdk2 and kinase-defective TETr-cdk2 (NI 32A) was confirmed byimmunoblot assay. Xenopus cyclin E (Jackson et al., 1995), like itshuman counterpart, also activated transcription in these assays (datanot shown). This activity was specific as xenopus cyclin E variants withpoint mutations affecting the cyclin box were inert.

[0263] The Second embodiment of the invention is useful to dynamicallyimage transcription in vivo. For example, cyclin E can activatetranscription under physiological conditions. 3T3 cells were transfectedwith the plasmid containing a selectable marker and TETo reporterplasmid with or without a plasmid encoding TETr-cdk2. Following drugselection, the stable transfectants were maintained as polyclonal poolsand serum starved into quiescence. At various timepoints after serumrefeeding, cell lysates were prepared and used in immunoblot, in vitrokinase, and luciferase assays (FIG. 14). In parallel, aliquots of thecells were analyzed for DNA content by FACS. In this system, S-phaseentry began 18-20 hours following the addition of serum. As expected,luciferase activity increased in the TETr-cdk2 producing cellscoincident with an increase in cyclin E protein levels and cyclinE-associated kinase activity (FIG. 14A-C). No such increase was observedin the cells producing equivalent amounts of TETr-cdk2 (N132A) ortransfected with the reporter alone (FIG. 14C and data not shown). Notethat the amount of TETr-cdk2 in these cells was less than the amount ofendogenous cdk2 (FIG. 14B). Thus, the results are unlikely to be anartifact of overproduction. Luciferase values declined as cyclin Elevels fell and cyclin A levels began to rise.

[0264] The Second embodiment of the invention in part relates to LGPscontaining cyclin-binding domains. Thus, these LGPs are useful todynamically quantify alterations in transcription of cell-cycleassociated genes, such as oncogenes and tumor suppressors. A LGPcontaining a cyclin-binding moiety can be localized to regionsundergoing cell proliferation, such as a tumor, and can be used toscreen for and determine the efficacy of cell proliferation-modulatingcompounds.

[0265] Materials and Methods

[0266] Cell Lines and Transfection

[0267] U2OS human osteosarcoma cells were grown in Dulbecco's modifiedEagle media (DMEM) supplemented with 10% heat-inactivated fetalclone(Hyclone) (FC), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2mM L-glutamine (PSG). SAOS-2 human osteosarcoma cells and NIH 3T3 mousefibroblast cells were grown in DMEM supplemented with 10%heat-inactivated fetal bovine serum (FBS) and PSG. NIH 3T3 stablesubclones transfected with the pCMV-neo and pUHC 13-3 reporter plasmidalone or with pSG5-TETr-cdk2 or with pSG5-TETr-cdk2(N132A) weremaintained in 0.7 mg/ml of G418. Cells were transfected using2×Bes-buffered saline (2×BBS)/calcium phosphate as described (Chen andOkayama, 1987). Where indicated doxycycline (Sigma) was added 24 hoursafter transfection to a final concentration of 2 μg/ml. Cells weremaintained in doxycycline for an additional 24 hours prior to harvest.

[0268] Plasmids

[0269] pRcCMV-cdk2 dominant negative (van den Heuvel and Harlow, 1993)was a gift of Dr. Ed Harlow; pVL1393-cdk2(N132A) (Xu et al., 1994) was agift of Dr. Helen Piwnica-Worms; pCD19 (Tedder and Isaacs, 1989) was agift of Dr. Thomas Tedder and pUHC13-3; ptet1-T81-luc, ptet2-T81-luc,ptet3-T81-luc, and ptet7-T81-luc (Gossen and Bujard, 1992) were gifts ofDr. Manfred Gossen. pSG5-TETr-E2F1, pSG5-TETr-RB, pSG5-HA-RB (Sellers,1995), pGEX-2TK-cdk2 (Adams et al., 1996) have been describedpreviously. To make pSG5-TETr-cyclin A, a protein phosphatase 1 (PP1)cDNA was first PCR amplified with oligos5′-GCGCTGATCAGGCGGAGGCGGATCAGGAGGAGGAGGATCAGGCGGAGGAGGATCAGGATCCATGTCCGACAGCGAGAA-3′ (SEQ ID NO: 7) and5′-GCGCGAATTCATTTCTTGGCTTTGGCAGA-3′ (SEQ ID NO: 8). The PCR product wascut with BclI and EcoRI and subcloned into pSG5-TETr cut with BamHI andEcoRI to make pSG5-TETr-(Gly4-Ser)₃-PP1. The cyclin A open reading frame(ORF) was PCR amplified with primers that introduced a 5′ BamHI site anda 3′ EcoRI site and subcloned into pSP72 (Promega) cut with these twoenzymes to make pSP72-cyclin A. The PP1 ‘stuffer’ frompSG5-TETr-(Gly4-Ser)₃—PP1 as then excised by digestion with BamHI andEcoRI and replaced with the cyclin A cDNA insert from pSP72-cyclin A. Tomake pSG5-TETr-cyclin E, the cyclin E ORF in pRcCMV-cyclin E was PCRamplified with primers that introduced a 5′ BglII and 3′ EcoRI site. ThePCR product was cut with these two enzymes and ligated into theBamHI-EcoRI backbone of pSG5-TETr-PP1. In parallel, these restrictedcyclin A and cyclin E PCR products were subcloned into pSG5-HA cut withBamHI and EcoRI to make pSG5-HA-cyclin A and pSG5-HA-cyclin E,respectively. Plasmids encoding cyclin A and cyclin E N-terminal andC-terminal deletion mutants were made in an analogous fashion by usingPCR primers that selectively amplified the desired coding regions. Tomake pSG5-TETr-cdk2 and pSG5-TETr-cdk2 (N132A), the cdk2 ORF inpRcCMV-cdk2 and pVL1393-cdk2 (N132A), respectively, were PCR amplifiedwith primers that introduced a 5′ BamHI and 3′ EcoRI site. The PCRproducts were cut with these two enzymes and ligated into theBamHI-EcoRI backbone of pSG5-TETr-PP1. All PCR reactions were performedwith Pfu DNA polymerase and the authenticity of plasmids containing theentire cyclin A, cyclin E, or cdk2 open reading frame was confirmed bydirect DNA sequencing. pSG5-TETr-cyclin A (E220A) and pSG5-TETr-cyclin E(L134A/Q174A) were generated using Transformer Site-Directed MutagenesisKit (Clontech) according to manufacture's instructions usingpSG5-TETr-cyclin A and pSG5-TETr-cyclin E as a template, respectively,and confirmed by DNA sequencing.

[0270] Antibodies and Immunoblot Analysis

[0271] Monoclonal anti-TETr was purchased from Clontech and anti-HA(12CA5) was purchased from Boehringer Mannheim. Polyclonal anti-cyclin A(SC-751), monoclonal and polyclonal anti-cyclin E (SC-247, SC-481), andpolyclonal anti-cdk2 (SC-163) were purchased from Santa Cruz. Cellextracts were made by lysis in EBC buffer (50 mM Tris [pH8], 120 mMNaCl, 0.5% Nonidet P-40). For immunoblot analysis, 100 μg of cellextract was loaded per lane. Nitrocellulose filters were blocked in 4%powdered milk/1% goat serum in TBS-T (10 mM Tris [pH 8], 0.05% Tween,150 mM NaCl) for 1 hour at room temperature prior to incubation inprimary antibody. Anti-HA (12CA5) was used at a concentration of 1.0μg/ml, anti-TETr antibody at 1:500 dilution (v/v), anti-cyclin A(SC-751) at 1:1,000 dilution (v/v), anti-cyclin E (SC-247, SC-481) at1:1,000 dilution (v/v) and anti-cdk2 (SC-163) at 1:1,000 dilution (v/v).Following 4 washes with TBS/T, bound antibody was detected usingalkaline phosphatase-conjugated secondary antibodies.

[0272] GST Pull-Down Assay

[0273] Glutathione S-transferase pull-down assays were performedbasically as described previously (Kaelin et al., 1991). Bindingreactions contained 10 μl of ³⁵S-radiolabelled in vitro translates madewith a TNT kit (Promega) and approximately 1 μg of the indicated GSTfusion protein in 1 ml of NETN (20 mM Tris [pH 8], 100 mM NaCl, 1 mMEDTA, 0.5% Nonidet P-40). Following 1 hour incubation at 4° C. withrocking, the Sepharose was washed 5 times with NETN. Bound proteins wereeluted by boiling in SDS-containing sample buffer and resolved bySDS-polyacrylamide gel electrophoresis. Comparable loading of GST-fusionproteins was confirmed by Coomassie brilliant blue staining and³⁵S-radiolabelled proteins were detected by fluorography.

[0274] FA CS/Cell Cycle Analysis

[0275] Fluorescence activated cell sorting (FACS) was done essentiallyas described (Qin et al., 1995). Briefly, subconfluent SAOS-2 cellsgrown in 100 mm dishes were transfected with 2 μg of pCD19 and 100 μg ofpSG5-HA-RB together with plasmids encoding the indicated cyclins. 72 hrlater the cells were harvested with trypsin-EDTA and stained withFITC-conjugated anti-CD19 antibody (CALTAG) and propiodium iodide.Samples were analyzed by two-color FACS with a FACScan (BectonDickinson). For cell cycle synchronization, cells were starved inserum-free DMEM for 72 hours before being stimulated with 10% FBS.

[0276] Luciferase Reporter Gene Assay

[0277] For TETr-fusion transcriptional assay, subconfluent U2OS cellswere transiently transfected in 6-well plates in duplicate with 1 μg ofpCMV-μgal, 1 μg of pUHC13-3 reporter plasmid, and 3 μg of the indicatedplasmids encoding TETr-fusion proteins. Sufficient parental pSG5-TETrwas added so that each reaction mix contained the same amount ofpSG5-TETr backbone. 48 hours after transfection luciferase activity andβ-galactosidase activity was determined as described previously (Qin,1995).

[0278] In Vitro Kinase Assay

[0279] 500 μg of cell extract was incubated with protein A Sepharose and1 μg of anti-cyclin E (SC-481) or anti-cyclin A (SC-751) antibody for 1hour at 4° C. in a final volume of 0.5 ml. The Sepharose was then washed5 times with NETN and 3 times in IP kinase (IPK) buffer (50 mM Tris-HCl[pH 7.5], 10 mM MgCl₂, 1 mM DTT). The Sepharose was then resuspended in27 μl of IPK buffer to which was added 2 μl of histone HI (1 mg/ml) and1 μl of [γ-³²P] ATP (6,000 Ci/mmol, 10 mCi/ml) and incubated for 30 minat 30° C. Reactions were stopped by addition of Laemmli sample buffer,boiled, resolved by SDS-polyacrylamidel gel electrophoresis, andsubjected to autoradiography.

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[0382] Equivalents

[0383] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference. The appropriate components, processes,and methods of those patents, applications and other documents may beselected for the present invention and embodiments thereof.

1 23 1 10 PRT Artificial Sequence Description of Artificial SequenceConsensus Target Peptide 1 Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp 1 510 2 12 PRT Artificial Sequence Description of Artificial SequenceTarget peptide 2 Gly Arg Pro Pro Val Lys Arg Arg Leu Asp Leu Glu 1 5 103 12 PRT Artificial Sequence Description of Artificial Sequence Targetpeptide 3 Cys Cys Ser Lys Ala Cys Arg Arg Leu Phe Gly Pro 1 5 10 4 8 PRTArtificial Sequence Description of Artificial Sequence Target peptide 4Trp Phe His Gly Lys Leu Ser Arg 1 5 5 8 PRT Artificial SequenceDescription of Artificial Sequence Target peptide 5 Trp Asn Val Gly SerSer Asn Arg 1 5 6 1210 PRT Artificial Sequence Description of ArtificialSequence Consensus Target Peptide 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 260 265 270 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 385 390 395 400 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 405 410 415 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa465 470 475 480 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 485 490 495 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 500 505 510 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 515 520 525 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 530 535 540 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 545 550 555 560 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 565 570 575 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 580 585 590 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Gly Ser Gly Ile Phe Leu Glu Thr 595 600 605 Ser Leu Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 610 615 620 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 625 630 635 640 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 645 650 655 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 660 665 670Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 675 680685 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 690695 700 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa705 710 715 720 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 725 730 735 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 740 745 750 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 755 760 765 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 770 775 780 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 785 790 795 800 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 805 810 815 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 820 825 830 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 835 840 845 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 850 855 860 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 865 870 875 880 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 885 890 895 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 900 905 910Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 915 920925 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 930935 940 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa945 950 955 960 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 965 970 975 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 980 985 990 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 995 1000 1005 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1010 1015 1020 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1025 1030 1035 1040 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1045 1050 1055 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1060 1065 1070 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1075 10801085 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1090 1095 1100 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 1105 1110 1115 1120 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 1125 1130 1135 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1140 1145 1150 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1155 1160 1165 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1170 1175 1180 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1185 1190 1195 1200Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1205 1210 7 78 DNA ArtificialSequence Description of Artificial Sequence PCR Primer 7 gcgctgatcaggcggaggcg gatcaggagg aggaggatca ggcggaggag gatcaggatc 60 catgtccgacagcgagaa 78 8 29 DNA Artificial Sequence Description of ArtificialSequence PCR Primer 8 gcgcgaattc atttcttggc tttggcaga 29 9 1206 PRTArtificial Sequence Description of Artificial Sequence Consensus TargetPeptide 9 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 245 250 255 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 385 390 395 400 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 405 410 415 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 465 470 475 480 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 485 490 495 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 500 505 510Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 515 520525 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 530535 540 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa545 550 555 560 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 565 570 575 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 580 585 590 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Leu LysXaa Leu Xaa Xaa 595 600 605 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 610 615 620 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 625 630 635 640 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 645 650 655 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 660 665 670 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 675 680 685 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 690 695 700 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 705 710 715 720 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 725 730 735 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 740 745 750Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 755 760765 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 770775 780 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa785 790 795 800 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 805 810 815 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 820 825 830 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 835 840 845 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 850 855 860 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 865 870 875 880 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 885 890 895 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 900 905 910 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 915 920 925 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 930 935 940 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 945 950 955 960 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 965 970 975 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 980 985 990Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 995 10001005 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1010 1015 1020 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 1025 1030 1035 1040 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 1045 1050 1055 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1060 1065 1070 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1075 1080 1085 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1090 1095 1100 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1105 1110 1115 1120Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 11251130 1135 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1140 1145 1150 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1155 1160 1165 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 1170 1175 1180 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1185 1190 1195 1200 Xaa Xaa Xaa Xaa Xaa Xaa1205 10 826 PRT Artificial Sequence Description of Artificial SequenceConsensus Target Peptide 10 Met Glu Gly Ala Gly Gly Ala Asn Asp Lys LysLys Ile Ser Ser Glu 1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala AlaArg Ser Arg Arg Ser Lys 20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala HisGln Leu Pro Leu Pro His 35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala SerVal Met Arg Leu Thr Ile 50 55 60 Ser Tyr Leu Arg Val Arg Lys Leu Leu AspAla Gly Asp Leu Asp Ile 65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met AsnCys Phe Tyr Leu Lys Ala Leu 85 90 95 Asp Gly Phe Val Met Val Leu Thr AspAsp Gly Asp Met Ile Tyr Ile 100 105 110 Ser Asp Asn Val Asn Lys Tyr MetGly Leu Thr Gln Phe Glu Leu Thr 115 120 125 Gly His Ser Val Phe Asp PheThr His Pro Cys Asp His Glu Glu Met 130 135 140 Arg Glu Met Leu Thr HisArg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr GlnArg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr 165 170 175 Ser Arg GlyArg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu 180 185 190 His CysThr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro 195 200 205 GlnCys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys 210 215 220Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu LeuGly 260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp HisLeu Thr 275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val ThrThr Gly Gln 290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val TrpVal Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn SerGln Pro Gln Cys Ile Val 325 330 335 Cys Val Asn Tyr Val Val Ser Gly IleIle Gln His Asp Leu Ile Phe 340 345 350 Ser Leu Gln Gln Thr Glu Cys ValLeu Lys Pro Val Glu Ser Ser Asp 355 360 365 Met Lys Met Thr Gln Leu PheThr Lys Val Glu Ser Glu Asp Thr Ser 370 375 380 Ser Leu Phe Asp Lys LeuLys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala AlaGly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn 405 410 415 Asp Thr GluThr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn 420 425 430 Asp ValMet Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu 435 440 445 AlaMet Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser 450 455 460Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp485 490 495 Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser ProGlu 500 505 510 Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser AspMet Val 515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe AlaGlu Asp Thr 530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr AspLeu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp AspAsp Phe Gln Leu Arg Ser 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu SerSer Ser Ala Ser Pro Glu Ser 580 585 590 Ala Ser Pro Gln Ser Thr Val ThrVal Phe Gln Gln Thr Gln Ile Gln 595 600 605 Glu Pro Thr Ala Asn Ala ThrThr Thr Thr Ala Thr Thr Asp Glu Leu 610 615 620 Lys Thr Val Thr Lys AspArg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser ProThr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser 645 650 655 Ser Pro TyrArg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala 660 665 670 Gly LysGly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro 675 680 685 AsnVal Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu 690 695 700Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu SerTrp 740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly MetGlu Gln 755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys ArgLeu Leu Gly 770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu ThrSer Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser ArgAsn Leu Leu Gln Gly Glu 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln ValAsn 820 825 11 1208 PRT Artificial Sequence Description of ArtificialSequence Consensus Binding Peptide 11 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 260 265 270 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 385 390 395 400 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 405 410 415Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 465 470 475 480 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 485 490 495 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 500 505 510 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 515 520 525 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 530 535 540 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 545 550 555 560 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 565 570 575 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 580 585 590 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Leu Xaa Pro Xaa Xaa Xaa Xaa 595 600 605 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 610 615 620 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 625 630 635 640 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 645 650 655Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 660 665670 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 675680 685 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa690 695 700 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 705 710 715 720 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 725 730 735 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 740 745 750 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 755 760 765 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 770 775 780 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 785 790 795 800 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 805 810 815 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 820 825 830 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 835 840 845 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 850 855 860 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 865 870 875 880 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 885 890 895Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 900 905910 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 915920 925 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa930 935 940 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 945 950 955 960 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 965 970 975 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 980 985 990 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 995 1000 1005 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 1010 1015 1020 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1025 1030 1035 1040 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1045 1050 1055 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1060 1065 1070Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 10751080 1085 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1090 1095 1100 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1105 1110 1115 1120 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1125 1130 1135 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1140 1145 1150 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1155 1160 1165 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1170 1175 1180 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1185 1190 11951200 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1205 12 8 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 12 Met Leu Ala ProTyr Ile Pro Met 1 5 13 8 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 13 Ala Leu Ala Pro Tyr Ile Pro Met1 5 14 8 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 14 Met Ala Ala Pro Tyr Ile Pro Met 1 5 15 8 PRTArtificial Sequence Description of Artificial Sequence synthetic peptide15 Met Leu Ala Ala Tyr Ile Pro Met 1 5 16 8 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 16 Met Leu Ala ProAla Ile Pro Met 1 5 17 8 PRT Artificial Sequence Description ofArtificial Sequence synthetic peptide 17 Met Leu Ala Pro Tyr Ala Pro Met1 5 18 8 PRT Artificial Sequence Description of Artificial Sequencesynthetic peptide 18 Met Leu Ala Pro Tyr Ile Ala Met 1 5 19 8 PRTArtificial Sequence Description of Artificial Sequence synthetic peptide19 Met Leu Ala Pro Tyr Ile Pro Ala 1 5 20 8 PRT Artificial SequenceDescription of Artificial Sequence synthetic peptide 20 Ala Ala Ala AlaAla Ala Ala Ala 1 5 21 8 PRT Artificial Sequence VARIANT (4) Wherein Xis a hydroxylated proline 21 Met Leu Ala Xaa Tyr Ile Pro Met 1 5 22 8PRT Artificial Sequence VARIANT (4) Wherein Xaa is hydroxylated proline22 Met Ala Ala Xaa Tyr Ile Pro Met 1 5 23 826 PRT Homo sapiens 23 MetGlu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu 1 5 10 15Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys 20 25 30Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His 35 40 45Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile 50 55 60Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile 65 70 7580 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu 85 9095 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile 100105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu GluMet 130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys GlyLys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met LysCys Thr Leu Thr 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser AlaThr Trp Lys Val Leu 180 185 190 His Cys Thr Gly His Ile His Val Tyr AspThr Asn Ser Asn Gln Pro 195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro MetThr Cys Leu Val Leu Ile Cys 210 215 220 Glu Pro Ile Pro His Pro Ser AsnIle Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg HisSer Leu Asp Met Lys Phe Ser Tyr Cys Asp 245 250 255 Glu Arg Ile Thr GluLeu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly 260 265 270 Arg Ser Ile TyrGlu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr 275 280 285 Lys Thr HisHis Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln 290 295 300 Tyr ArgMet Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val 325 330335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe 340345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp ThrSer 370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu ThrLeu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu AspPhe Gly Ser Asn 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu GluVal Pro Leu Tyr Asn 420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu LysLeu Gln Asn Ile Asn Leu 435 440 445 Ala Met Ser Pro Leu Pro Thr Ala GluThr Pro Lys Pro Leu Arg Ser 450 455 460 Ser Ala Asp Pro Ala Leu Asn GlnGlu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu GluLeu Ser Phe Thr Met Pro Gln Ile Gln Asp 485 490 495 Gln Thr Pro Ser ProSer Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu 500 505 510 Pro Asn Ser ProSer Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val 515 520 525 Asn Glu PheLys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr 530 535 540 Glu AlaLys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser 565 570575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser 580585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp GluLeu 610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile LeuIle Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr ThrSer Ala Thr Ser 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr AlaSer Pro Asn Arg Ala 660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu LysSer His Pro Arg Ser Pro 675 680 685 Asn Val Leu Ser Val Ala Leu Ser GlnArg Thr Thr Val Pro Glu Glu 690 695 700 Glu Leu Asn Pro Lys Ile Leu AlaLeu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp GlySer Leu Phe Gln Ala Val Gly Ile Gly Thr 725 730 735 Leu Leu Gln Gln ProAsp Asp His Ala Ala Thr Thr Ser Leu Ser Trp 740 745 750 Lys Arg Val LysGly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln 755 760 765 Lys Thr IleIle Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly 770 775 780 Gln SerMet Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu 805 810815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn 820 825

What is claimed is:
 1. A method of identifying prolyl hydroxylasemodulators, comprising the steps of: a) contacting a prolyl hydroxylase,a putative prolyl hydroxylase modulator and a light-generating fusionprotein comprising an HIFα polypeptide moiety having binding characterfor prolyl hydroxylase and a light-generating polypeptide moiety,wherein the light generation of said light-generating polypeptide moietychanges upon binding of a prolyl hydroxylase to said HIFα polypeptidemoiety, under conditions favorable for binding of a prolyl hydroxylaseto a HIFα to form a test sample; and b) determining the ability of saidputative prolyl hydroxylase modulator to modulate prolyl hydroxylase bymeasuring the light generated in said test sample.
 2. An assay for amodulator of prolyl hydroxylase, comprising a) bringing into contact aprolyl hydroxylase, a HIF1α polypeptide moiety and a putative modulatorcompound under conditions where prolyl hydroxylase, in the absence ofmodulator, is capable of acting on said HIF1α polypeptide moiety; and b)measuring the degree of prolyl hydroxylase inhibition caused by saidmodulator compound.
 3. The method of claim 2, wherein said test samplefurther comprises pVHL.
 4. The method of claim 2, wherein said HIF1αpolypeptide moiety comprises the amino acid sequenceY-X₁-Leu-X₂-Pro_(h)-X₃-X₄-X₅-X₆-Y′, wherein a) Pro_(h) is hydroxylatedproline; b) X_(1,) X_(2,) X_(4,) X_(5,) and X₆ are independently Gly,Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp; c) X₃ is Ser, Thr, or Tyr;and d) Y and Y′ are independently present or absent and, if present,independently comprise a peptide having from 1 to 600 amino acids. 5.The method of claim 2, wherein said HIF1α polypeptide moiety comprisesthe amino acid sequence corresponding to the N-terminal residues 1-600of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue564 is hydroxylated proline.
 6. The method of claim 2, wherein saidHIF1α polypeptide moiety comprises the amino acid sequence correspondingto the N-terminal residues 1-600 of HIF1α, numbered in accordance withwild-type HIF1α, wherein residue 402 is hydroxylated proline.
 7. Themethod of claim 2, wherein said HIF1α polypeptide moiety comprises theamino acid sequence corresponding to the N-terminal residues 1-600 ofHIF1α, numbered in accordance with wild-type HIF1α, wherein either orboth of residues 402 and 564 are hydroxylated proline.
 8. The method ofclaim 2, wherein said HIF1α polypeptide moiety comprises a 4 to 12 aminoacid sequence corresponding to the residues adjacent to and/orsurrounding residue 564, inclusive, of HIF1α, numbered in accordancewith wild-type HIF1α, wherein residue 564 is hydroxylated proline. 9.The method of claim 2, wherein said HIF1α polypeptide moiety comprises a12 to 14 amino acid sequence corresponding to the residues adjacent toand/or surrounding residue 564, inclusive, of HIF1α, numbered inaccordance with wild-type HIF1α, wherein residue 564 is hydroxylatedproline.
 10. The method of claim 2, wherein said HIF1α polypeptidemoiety comprises a 20 to 30 amino acid sequence corresponding to theresidues adjacent to and/or surrounding residue 564, inclusive, ofHIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564is hydroxylated proline.
 11. The method of claim 2, wherein said HIF1αpolypeptide moiety comprises a 80 to 120 amino acid sequencecorresponding to the residues adjacent to and/or surrounding residue564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α,wherein residue 564 is hydroxylated proline.
 12. The method of claim 1,whereupon ligand binding to said ligand binding site alters the lightgeneration of said light-generating fusion protein without altering thephosphorylational state of said light-generating fusion protein.
 13. Themethod of claim 2, wherein said HIF1α polypeptide moiety comprises aminoacids 555-575 of wild type HIFα, numbered in accordance with wild typeHIFα.
 14. HIFα polypeptide comprising amino acids 555-575 of wild typeHIFα, numbered in accordance with wild type HIFα.
 15. A method oftreating or preventing a hypoxic or ischemic related disorder in asubject, comprising administering to a subject in need thereof acompound identified by the method of claim 1 which decreases prolylhydroxylase expression or activity, such that said hypoxic or ischemicrelated disorder is treated.
 16. The method of claim 15, wherein saidcompound is a prolyl hydroxylase antibody, or a nucleic acid thatdecreases the expression of a nucleic acid that encodes a prolylhydroxylase polypeptide.
 17. The method of claim 16, wherein the nucleicacid is a prolyl hydroxylase anti-sense nucleic acid.
 18. The method ofclaim 15, wherein the hypoxic or ischemic related disorder is an acuteevent selected from the consisting of myocardial infarction, stroke,cancer, and diabetes.
 19. The method of claim 15, wherein the hypoxic orischemic related disorder is a chronic event not caused by tissuescarring.
 20. The method of claim 15, wherein the hypoxic or ischemicrelated disorder is a chronic event selected from the group consistingof deep vein thrombosis, pulmonary embolus, and renal failure.
 21. Themethod of claim 15, wherein the half life of HIF in said subject isincreased compared to a subject not exposed to said compound.
 22. Amethod of increasing angiogenesis or vascularization in a subject,comprising administering to a subject in need thereof a compoundidentified by the method of claim 1 which decreases prolyl hydroxylaseexpression or activity.
 23. A method of treating cancer in a subject,comprising administering to a subject in need thereof a compoundidentified by the method of claim 1 which increases prolyl hydroxylaseexpression or activity.
 24. A method of treating a cell-proliferatingdisorder by administering to a subject an effective amount of a fusionprotein comprising a HIFα polypeptide moiety having a binding characterfor prolyl hydroxylase, and a suicide polypeptide moiety, such that saidcell-proliferating disorder is treated.
 25. A method of treating ahypoxic or ischemic disorder by administering to a subject an effectiveamount of a fusion protein comprising a HIFα polypeptide moiety having abinding character for prolyl hydroxylase, and a suicide polypeptidemoiety, such that said hypoxic or ischemic disorder is treated.
 26. Amethod of killing hypoxic tumor cells, comprising administration of aneffective amount of a fusion protein to a subject, said fusion proteincomprising a HIFα polypeptide moiety having a binding character forprolyl hydroxylase, and a suicide polypeptide moiety, such that saidhypoxic tumor cells are killed.
 27. A method of monitoring the treatmentof a cell-proliferating disorder by a) administering to a subject aneffective amount of a fusion protein comprising a HIFα polypeptidemoiety having a binding character for prolyl hydroxylase, a suicidepolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of said light-generating fusion protein changesupon binding of prolyl hydroxylase to said HIFα polypeptide moiety, suchthat said cell-proliferating disorder is treated, and b) monitoring theability of said fusion protein to inhibit cell proliferation bymeasuring the light generated by said light-generating fusion protein.28. A method of treating a cell-proliferating disorder by administeringto a subject an effective amount of a fusion protein comprising acyclin/cdk binding site and a suicide protein polypeptide moiety, suchthat said cell-proliferating disorder is treated.
 29. A method ofmonitoring the treatment of a cell-proliferating disorder by a)administering to a subject an effective amount of a light-generatingfusion protein comprising a cyclin/cdk binding site, a suicide proteinpolypeptide moiety, and a light-generating polypeptide moiety, whereinthe light generation of said light-generating fusion protein changesupon binding of a cyclin to said cyclin/cdk binding site, such that saidcell-proliferating disorder is treated; and b) monitoring the ability ofsaid fusion protein to inhibit cell proliferation by measuring the lightgenerated by said light-generating fusion protein.
 30. A method ofscreening candidate compounds that inhibit cell proliferation,comprising the steps of: a) contacting cells with a candidate compoundand a light-generating fusion protein comprising a cyclin/cdk bindingsite and a light-generating polypeptide moiety, wherein the lightgeneration of said light-generating fusion protein changes upon bindingof a cyclin at said cyclin binding site, said cyclin binding indicativeof cancerous tissue; and b) determining the ability of said candidatecompound to inhibit cell proliferation by measuring the luminescence ofsaid cells.
 31. A cell proliferation-inhibiting compound identified bythe method of claim
 30. 32. A method for screening for a modulator ofactivity or latency of, or predisposition to a disorder, said methodcomprising: a) administering a test compound to a test animal atincreased risk for a disorder, wherein said test animal recombinantlyexpresses a light-generating fusion protein comprising a ligand bindingsite and a light-generating polypeptide moiety, wherein the lightgeneration of said light-generating fusion protein changes upon bindingof a ligand at said ligand binding site, said ligand binding siterecognizing a ligand on an entity associated with a disorder, or aproduct of said disorder; b) allowing for localization of saidlight-generating fusion protein and an entity, wherein contact betweensaid ligand binding site and a ligand associated with said disordercauses a modification of a colinear effector site which alters the lightgeneration of said light-generating polypeptide moiety; c) detecting theluminescence of said light-generating polypeptide moiety in said testanimal after administering the compound of step (a); and d) comparingthe luminescence of said light-generating polypeptide moiety in saidtest animal with the luminescence of said light-generating polypeptidemoiety in a control animal not administered said compound, wherein achange in the activity of said light-generating polypeptide moiety insaid test animal relative to said control animal indicates the testcompound is a modulator of latency of or predisposition to, a disorder.33. The method of claim 32, wherein the disorder is a hypoxia-relateddisorder.
 34. The method of claim 32, wherein the disorder is selectedfrom the group consisting of cancer, diabetes, heart disease and stroke.35. A non-invasive method for determining the effects of an anti-hypoxiccompound in vivo, comprising: a) administering to a subject alight-generating fusion protein, or a cell expressing saidlight-generating fusion protein, comprising an ubiquitin ligase bindingsite and a light-generating polypeptide moiety, wherein the lightgeneration of said light-generating fusion protein changes upon bindingof a ubiquitin ligase at said ubiquitin ligase binding site, saidubiquitin ligase binding site recognizing a ubiquitin ligase present inhypoxic conditions in hypoxic tissue; b) allowing for localization ofsaid light-generating fusion protein or cell in hypoxic tissue in saidsubject, wherein contact between said ubiquitin ligase binding site anda ubiquitin ligase causes a modification of a colinear effector sitewhich alters the light generation of said light-generating polypeptidemoiety; and; c) determining the ability of said candidate compound toinhibit hypoxia by measuring the luminescence of said localizedlight-generating fusion protein.
 36. The method of claim 35, whereinsaid ligand binding site comprises the amino acid sequenceY-X₁-Leu-X₂-Pro_(h)-X₃-X₄-X₅-X₆-Y′, wherein a) Pro_(h) is hydroxylatedproline; b) X_(1,) X_(2,) X_(4,) X_(5,) and X₆ are independently Gly,Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp; c) X₃ is Ser, Thr, or Tyr;and d) Y and Y′ are independently present or absent and, if present,independently comprise a peptide having from 1 to 600 amino acids. 37.The method of claim 35, wherein said ligand binding site comprises theamino acid sequence corresponding to the N-terminal residues 1-600 ofHIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564is hydroxylated proline.
 38. The method of claim 35, wherein said ligandbinding site comprises the amino acid sequence corresponding to theN-terminal residues 1-600 of HIF1α, numbered in accordance withwild-type HIF1α, wherein residue 402 is hydroxylated proline.
 39. Themethod of claim 35, wherein said ligand binding site comprises the aminoacid sequence corresponding to the N-terminal residues 1-600 of HIF1α,numbered in accordance with wild-type HIF1α, wherein either or both ofresidues 402 and 564 are hydroxylated proline.
 40. The method of claim35, wherein said ligand binding site comprises a 4 to 12 amino acidsequence corresponding to the residues adjacent to and/or surroundingresidue 564, inclusive, of HIF1α, numbered in accordance with wild-typeHIF1α, wherein residue 564 is hydroxylated proline.
 41. The method ofclaim 35, wherein said ligand binding site comprises a 12 to 14 aminoacid sequence corresponding to the residues adjacent to and/orsurrounding residue 564, inclusive, of HIF1α, numbered in accordancewith wild-type HIF1α, wherein residue 564 is hydroxylated proline. 42.The method of claim 35, wherein said ligand binding site comprises a 20to 30 amino acid sequence corresponding to the residues adjacent toand/or surrounding residue 564, inclusive, of HIF1α, numbered inaccordance with wild-type HIF1α, wherein residue 564 is hydroxylatedproline.
 43. The method of claim 35, wherein said ligand binding sitecomprises a 80 to 120 amino acid sequence corresponding to the residuesadjacent to and/or surrounding residue 564, inclusive, of HIF1α,numbered in accordance with wild-type HIF1α, wherein residue 564 ishydroxylated proline.
 44. A noninvasive method for detecting the effectsof an anti-cell proliferation compound under study in a mammaliansubject, comprising: a) administering to a subject a light-generatingfusion protein, or a cell expressing said light-generating fusionprotein, comprising a cyclin/cdk binding site and a light-generatingpolypeptide moiety, wherein the light generation of saidlight-generating fusion protein changes upon binding of a cyclin at saidcyclin binding site, said cyclin binding indicative ofcell-proliferating tissue; b) allowing for localization of saidlight-generating fusion protein or cell in cell-proliferating tissue insaid subject, wherein contact between said cyclin binding site and acyclin causes a modification of a colinear effector site which altersthe light generation of said light-generating polypeptide moiety; and c)determining the ability of said candidate compound to inhibit cellproliferation by measuring the luminescence from said localizedlight-generating fusion protein.
 45. A method of treating or preventinga hypoxic or ischemic related disorder in a subject, comprisingadministering to a subject in need thereof a compound which modulatesprolyl hydroxylation of HIF, such that said hypoxic or ischemic relateddisorder is treated.
 46. The method of claim 46, wherein said compounddecreases prolyl hydroxylation of HIF.
 47. The method of claim 46,wherein said compound increases prolyl hydroxylation of HIF.
 48. Amethod of regulating HIF turnover in a subject, comprising administeringto a subject in need thereof a compound which modulates prolylhydroxylation of HIF, such that HIF turnover is regulated.
 49. Themethod of claim 49, wherein said compound increases prolyl hydroxylationof HIF.
 50. The method of claim 49, wherein said compound decreasesprolyl hydroxylation of HIF.
 51. A method of treating or preventing aHIF-related disorder in a subject, comprising administering to a subjectin need thereof a compound which modulates prolyl hydroxylation of HIF,such that said HIF-related disorder is prevented, reversed orstabilized.
 52. The method of claim 52, wherein said compound increasesprolyl hydroxylation of HIF.
 53. The method of claim 52, wherein saidcompound decreases prolyl hydroxylation of HIF.
 54. The method of claim52, wherein said HIF-related disorder is selected from the groupconsisting of myocardial infarction, stroke, cancer, and diabetes.